Highly selective nucleic acid amplification primers

ABSTRACT

This invention discloses multi-part primers for primer-dependent nucleic acid amplification methods. Also disclosed are primer-dependent nucleic acid amplification reactions, particularly DNA amplification reactions, reaction mixtures and reagent kits for such reactions. This invention relates to primer-dependent nucleic acid amplification reactions, particularly DNA amplification reactions such as PCR, and primers, reaction mixtures and reagent kits for such reactions and assays employing same.

CROSS REFERENCE TO RELATED APPLICATION

This application claims priority of U.S. Provisional Application No.61/762,117 filed on Feb. 7, 2013. The content of the application isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to primer-dependent nucleic acid amplificationreactions, particularly DNA amplification reactions such as PCR, andprimers, reaction mixtures and reagent kits for such reactions andassays employing same.

BACKGROUND OF THE INVENTION

Primer-dependent nucleic acid amplification reactions, which may includedetection of amplification products (“amplicons”), require“specificity,” that is, annealing of a primer to the intended place in anucleic acid strand and extension of primers bound only to the intendedtarget sequence. Conventionally, specificity is obtained by making aprimer sufficiently long so that under the amplification reactionconditions, primarily during the primer-annealing step, the primer goesto only one place in a nucleic acid strand.

Certain amplification reactions are intended to distinguish between oramong allelic variants, for example, single-nucleotide polymorphisms(SNPs). One way to do that is to amplify all variants and to distinguishbetween or among them by allele-specific hybridization probes such asmolecular beacon probes. For such an approach, the amplification primersare made equally complementary to all variants so as to amplify a regionthat includes the sequence that varies between or among alleles, and aprobe identifies an allele that is present in the amplified product orproducts. See, for example, Tyagi et al. (1998) Nature Biotechnology16:49-53. If the sequence being investigated is an allele, such as a SNPthat is present in a mixture with another allele, for example, awild-type (WT) variant, distinguishing by use of a probe has a practicaldetection limit of about 3% (not less than about 30,000 target allelemolecules in the presence of 1,000,000 molecules of the alternateallele) due to the tendency of amplification of the prevalent allele tooverwhelm amplification of the rare allele.

Another way to distinguish between or among alleles is to use a primerthat is selective for the sequence being investigated. For such anapproach, the primer is made complementary to the sequence that variesbetween or among alleles, and amplified product may be detected eitherby labeled primers, a DNA binding dye, or a labeled probe (in this casethe probe detects a sequence common to amplicons of all alleles). Aprimer that is highly specific typically has a length of 15-30nucleotides. Such a conventional primer has very limited selectivity forone allele over another. It is known that shortening a primer willimprove its selectivity, but because that improvement comes at theexpense of specificity, and because short primers are unlikely to formstable hybrids with their target sequence at typical annealingtemperatures, shortening a primer is of limited value for analyzingmixtures of alleles.

Other modifications of primers have been developed to improve theirselectivity while retaining specificity. One such approach is ARMS(“amplification refractory mutation system”). An ARMS primer has a3′-terminal nucleotide that is complementary to the sequence variantbeing investigated, but that is mismatched to another allele or alleles.See Newton et al. (1989) Nucleic Acids Res. 17:2503-2516; and Ferrie etal. (1992) Am. J. Hum. Genet. 51:251-262. ARMS relies on the refractorynature of certain DNA polymerases, that is, a tendency not to extend aprimer-target hybrid having such a mismatch. ARMS has been demonstratedto be useful for determining zygosity (homozygous WT, heterozygous, orhomozygous mutant (MUT)), but it has a practical detection limit forother uses of about 1% (not less than about 10,000 target allelemolecules in the presence of 1,000,000 molecules of the alternateallele).

Another approach is to make a primer into a hairpin to increase itsselectivity. See Tyagi et al. European patent EP 1 185 546 (2008), whichdiscloses making the hairpin loop complementary to the sequence beinginvestigated but mismatched to another allele or alleles; and Hazbon andAlland (2004) J. Clin. Microbiol. 42:1236-1242, which discloses makingthe terminal nucleotide of the 3′ arm of the hairpin primercomplementary to the sequence variant being investigated but that ismismatched to another allele or alleles, as with ARMS. Thesemodifications also have practical detection limits of about 1% (not lessthan about 10,000 target allele molecules in the presence of 1,000,000molecules of the alternate allele).

Jong-Yoon Chun and his colleagues at the Seegene Institute of LifeScience in Seoul, South Korea, have devised a type of primer that theyrefer to as a “dual-priming oligonucleotide (DPO).” See, Chun et al.(2007) Nucleic Acids Res. 35 (6) e40; Kim et al. (2008) J. Virol. Meth.149:76-84; Horii et al. (2009) Lett. Appl. Microbiol. 49:46-52; WO2006/095981 A1; and WO 2007/097582 A1. A DPO primer consists of threesegments: a long 5′ high-temperature segment, for example, 20-25nucleotides in length, a central separation segment of fivedeoxyriboinosines, and a 3′ priming segment, generally 8-12 nucleotidesin length, that is complementary to the intended target sequence butmismatched to other target sequences. The target sequence iscomplementary to all three segments, but the Tm of the 3′ segment islower than the Tm of the 5′ segment, due to its shorter length, and theseparation segment has the lowest Tm due to the five deoxyriboinosines.A DPO primer is designed such that amplification results only if boththe 5′ segment and the 3′ segment hybridize to a target strand.According to Chun et al. (2007), the separation segment was selected tobe five deoxyriboinosines, because 3-4 and 6-8 deoxyriboinosines did notgive results as good; the 3′ segment was positioned so as to provide aGC content of 40-80%, and the 5′ segment was provided a lengthsufficient to raise its Tm above the annealing temperature to be used in3′-RACE amplifications (Nucleic Acids Res. 35(6) e40 at page 2). Chun etal. reports successful genotyping (homozygous wild type, heterozygous,or homozygous mutant) of a SNP (G→A mutation) in the CYP2C19 gene usingtwo pairs of DPO primers. Of the four DPO primers, one had a 3′ segment12-nucleotides long, perfectly complementary to both alleles; one had a3′ segment 9-nucleotides long, perfectly complementary to both alleles;and two had 3′ segments 8-nucleotides long with the variable nucleotidelocated in the middle, that is, at the fourth nucleotide position fromthe 3′ end. Genotyping was accomplished by means of gel electrophoresis.

There are situations in which it is desired to detect a very rare firstallele in the presence of a very abundant second allele. This has beentermed “sensitivity”. In other words, the primer must not only be“specific” (go to the correct place in the genome), and be “selective”(reject wild type or other abundant sequences similar to the targetsequence), but it must be highly selective, that is, “sensitive” enoughto detect a very few mutant or other rare first sequence in the presenceof an abundance of wild type or other abundant second sequence. SeeMakarov and Chupreta international patent application WO 2012/112 582 A2at paragraph [0004].

To improve sensitivity while retaining specificity and selectivity,Vladimir Makarov and his colleagues at Swift Biosciences (Ann Arbor,Mich., U.S.A.) disclose a “discontinuous polynucleotide [“primer”]design” (WO 2012/112 582 A2 at paragraph [0051]) that has beencommercialized as myT™ Primers. Such primers may be viewed as longconventional primers that are composed of two oligonucleotides so as tocreate an eight-nucleotide 3′ priming sequence; and adding complementarytails to the 5′ end of that sequence and to the 3′ end of the otheroligonucleotide to form a high-temperature stem. Through the stem, thetwo oligonucleotides are joined non-covalently and form a stablethree-way junction when bound to the target sequence. Theoligonucleotide with the eight-nucleotide 3′ end is referred to as the“primer”, and the other oligonucleotide is referred to as the “fixer”.The function of the fixer is to provide specificity, that is, to bindthe primer to the intended place in the genome. It is accordingly long,typically about 30-nucleotides in length. The function of the tails isto hybridize the two oligonucleotides under amplification conditions, sothe tails also are fairly long, forming a stem 20-25 nucleotides inlength. The function of the eight-nucleotide 3′ region is to prime withselectivity. The discontinuous hybridization “in effect stabilizedbinding between the [priming] region of the primer oligonucleotide evenif this region is as small as eight bases, thereby increasing theefficiency of PCR.” (WO 2012/112582 A2). Further improvements aredisclosed in Examples 9-11 of WO 2012/112582 A2. The nucleotide that ismismatched to the wild-type target is made the 3′-terminal nucleotide,as in ARMS; a third oligonucleotide, a blocking oligonucleotide(“blocker”), whose 5′-terminal nucleotide overlaps the 3′-terminalnucleotide of the primer and is complementary to the wild-type target,is included in the amplification reaction; and the 3′-terminalnucleotide of the primer is made of locked nucleic acid (“LNA”). For thedetection of single-nucleotide polymorphisms in the K-ras and B-rafgenes, detection sensitivity of one mutant in 14,000 wild-type(approximately 0.01%) was disclosed.

There remains a need for a single-oligonucleotide primer that has theability to detect and, preferably, to quantify the number of a rarefirst target sequence, for example, a mutant target sequence, in thepresence of a very large number of a second target sequence that differsfrom the first target sequence by as little as a single nucleotide, forexample, a wild-type sequence.

SUMMARY OF INVENTION

This invention includes a multi-part primer for primer-dependent nucleicacid amplification methods, including particularly polymerase chainreaction (PCR) methods, that is capable of distinguishing between a rareintended target (e.g., a mutant DNA target) and a closely relatedsequence (e.g., a wild-type DNA target) that differs by asingle-nucleotide substitution, sometimes referred to as asingle-nucleotide polymorphism, for short, a SNP.

This invention includes primer-dependent nucleic acid amplificationmethods, for example PCR methods, that utilize a multi-part primeraccording to this invention and that are capable of selectivelyamplifying one or more rare target sequences in a population of abundantclosely related sequences. Such intended target sequences may be raremutant sequences, for example, sequences found in malignant cells, in anotherwise abundant wild-type population found in normal cells. Formethods such as PCR methods that utilize a DNA-dependent DNA polymerase,the intended target and related sequences are DNA sequences that occurin a sample, or they are cDNA sequences that are made by reversetranscription from RNA sequences, including mRNA sequences, that occurin a sample. Reverse transcription may be performed in the same reactionmixture as subsequent amplification, or it may be performed separatelybefore amplification. Multi-part primers can be used as primers inreverse transcription reactions. This invention also includesamplification and detection methods that include detection of amplifiedproducts, or “amplicons”. The description that follows, including theExample, describes multi-part primers in connection with PCRamplification reactions starting with DNA targets. Persons skilled inthe art will understand how to apply these teachings to multi-partprimers in connection with other primer-dependent nucleic acidamplification methods.

This invention further includes reagent kits containing reagents forperforming such amplification methods, including such amplification anddetection methods.

This invention addresses, inter alia, a major goal of moleculardiagnostics, which is to find a sensitive and specific means fordetecting extremely rare cancer cells (by virtue of an identifyingsomatic mutation) in a clinical sample containing very abundant normalcells, and to be able to quantitatively determine their abundance. Thereare multiple advantages of being able to do this, including:

1. The ability to detect the presence and abundance of cancer cellsafter treatment (such as after a bone marrow transplant in leukemiapatients). Utilizing this invention will enable physicians to determinewhether the administration of (rather toxic) drugs can be discontinued.This invention will enable clinical studies to be carried out todetermine the level of minimum residual disease that can be handled bythe body without drug treatment. Moreover, patients can be monitoredover time after treatment to detect the appearance of higher levels thatcan then be treated by appropriate means.

2. The ability to rapidly detect and quantitate rare cancer cells inbiopsies taken during surgery (at levels too low to be seen in amicroscope by a pathologist). Utilizing this invention will enablesurgeons to rationally decide the extent of surgery, sparing the removalof unaffected tissues.

3. The ability to detect key mutations in DNA molecules released intoblood plasma by the natural process of destruction of rare circulatingtumor cells in blood. Utilizing this invention will enable the earlydetection of tumors whose cells have acquired the ability tometastasize, providing physicians an opportunity for early intervention.

4. The ability to monitor patients whose genetic inheritance suggeststhat life-threatening tumors can arise during their lifetime (such as inmany breast cancers). Utilizing this invention will enable periodicmonitoring to determine if key somatic mutations have occurred, so thattherapeutic intervention can be provided at a very early stage in thedisease.

Other applications for this invention will occur to persons skilled inthe art.

By “rare” and “abundant” is meant that the ratio of intended targetsequences to closely related sequences is at least in the range of 1/10³to 1/10⁷ (that is, one in a thousand, one in ten thousand, one inone-hundred thousand, one in a million, or one in ten million). By“closely related” is meant a sequence that differs from an intendedtarget sequence by one, two, or at most a few nucleotides. Mutant targetsequences that differ from wild-type sequences at a particular locationby a single nucleotide are commonly referred to as being or having asingle-nucleotide polymorphism (SNP).

Methods according to this invention include primer-dependent nucleicacid amplification for at least one intended target sequence (e.g., amutant DNA target sequence), which may occur rarely in a sample orreaction mixture containing an abundance of the closely related,unintended target sequence (e.g., a wild-type DNA target sequence).These methods utilize a reaction mixture that contains for each raretarget a multi-part primer according to this invention. Three parts ofthe primer cooperate with one another to yield an amplification that isextremely selective. FIG. 1 is a schematic representation of a primeraccording to this invention. FIG. 1 includes two schematics: the topschematic shows a multi-part primer 103 under hybridization conditions,such as occurs during the annealing step of a PCR cycle, in relation toits intended target 101, which may be rare; and the bottom schematicshows the same primer in relation to a closely related sequence, hereinreferred to as an unintended or mismatched target 102. Intended target101 and unintended target 102 have the same nucleotide sequence, exceptthat intended target 101 has one or more nucleotides “x”, preferably asingle nucleotide, that differ from the corresponding nucleotide ornucleotides in mismatched target 102, here designated “y”. For example,unintended target sequence 102 may be a wild-type human DNA sequence,and intended target sequence 101 may be a mutant cancer cell sequencecontaining a SNP. The upper schematic depicts a primer 103 that ishybridized to intended target strand 101. In the 5′-to-3′ direction, theprimer includes anchor sequence 104, bridge sequence 105, and footsequence 106. Primer 103 optionally may include a 5′ tail 107 to impartadded functionality. It also optionally includes a blocking group 108.During primer annealing at the start of amplification, anchor sequence104 hybridizes to intended target 101, as conventionally indicated bythe short vertical lines between the anchor sequence and its bindingsite (representing the pairing of complementary nucleotides). Bridgesequence 105 is mismatched (not complementary) to target 101 at sequence109, which we refer to as the “intervening sequence,” and causes a“bubble” in the duplex structure. Foot sequence 106 hybridizes tointended target 101 and primes copying by a DNA polymerase. The lowerschematic depicts the same primer 103 that is hybridized to unintended,mismatched target 102. As stated, mismatched target 102 differs fromintended target 101 by at least one nucleotide change (x to y) in thesequence opposite primer foot 106. During primer annealing at the startof amplification, anchor sequence 104 hybridizes to unintended target102 at the anchor-sequence binding site, as shown. Again, bridgesequence 105 is mismatched to intervening sequence 109. However, footsequence 106 is not hybridized to target 102, and target 102 is notprimed for copying.

In an ideal amplification reaction according to FIG. 1, intended target101, even if rare, would always be copied, and unintended target 102,even if abundant, would never be copied. However, priming is astatistical matter. For example, primers go on and off targets, perfectand mismatched, with some frequency. Consequently, perfect targets arenot always copied, and mismatched targets are sometimes copied.Selectively amplifying and detecting rare targets thus depends both onthe frequency at which perfect targets are copied and on the frequencyat which mismatched targets are copied. Multi-part primers useful inthis invention have three contiguous sequences (anchor sequence, bridgesequence and foot sequence) that cooperate with one another to achievevery high selectivity in practical amplification reactions, includingamplification-and-detection assays. The anchor sequence serves tohybridize the primer to the target sequence, which is the same (oralmost the same) in the intended target and the unintended, mismatchedtarget, in an efficient manner not dissimilar to hybridization of aconventional primer. The bridge and foot sequences, more fully describedbelow, cooperate to impart primer specificity, that is, selectivity forthe intended target over the mismatched target. We have discovered thata high degree of selectivity is achieved if the bridge and footsequences cooperate to make copying of the intended target unlikelyrather than likely. Further, we make the bridge sequence rabidly andefficiently copyable. The bridge sequence is preferably a DNA sequence.The result achieved is amplification of the intended target sequencethat is delayed in starting, but that proceeds normally once it hasbegun; but amplification of the unintended, mismatched target sequencethat is significantly more delayed but that proceeds normally once ithas begun. The increased delay for the mismatched target relative to thematched target is an improvement in selectivity achieved by the primer.Such improved selectivity is achieved, because the probability of theunintended target sequence being copied by a DNA polymerase is at least1,000 times less than the probability of the intended target sequencebeing copied, preferably at least 10,000 times less and more preferablyat least 100,000 times less.

Referring to FIG. 1, the primer includes an anchor sequence 104 thathybridizes the primer to a binding site in the intended target and theclosely related target sequence during the primer-annealing step, whichincludes a primer-annealing temperature, of the amplification reaction.In that regard, the anchor sequence is like, and functions like, aconventional primer. It may be perfectly complementary to the target andto the closely related sequence, or it may contain one or moremismatched nucleotides. In the amplification reaction in which it isused, it generally has a melting temperature, Tm, at least equal to orabove the annealing temperature, so as to enhance hybridization. In mostof the Examples the anchor sequence Tm is between 3° C. and 10° C. abovethe primer-annealing temperature. To the extent not prevented by ablocking group, all or a portion of anchor sequences of multi-partprimers used in this invention are copied by DNA polymerase. Becauseexponential amplification proceeds rapidly with high, normal PCRefficiency, the inclusion of non-natural nucleotides, nucleotide mimics,and non-natural internucleotide linkages in copied portions is limitedto types and numbers that permit rapid and efficient copying by DNApolymerase. We prefer that anchor sequences be DNA sequences.

Anchor sequence 104 typically forms a probe-target hybrid 15-40nucleotides in length, preferably 15-30 nucleotides in length, and morepreferably 20-30 nucleotides in length. Shorter anchor sequences muststill hybridize to their target sequences during primer annealing, asstated above, which often means that their Tm's must be at least 50° C.(e.g., 66-72° C.). It may be perfectly complementary to the target, orit may contain one or more mismatches; for example, where one isinvestigating a target whose sequence versus the anchor is variable, onemay choose an anchor sequence 104 that is a consensus sequence that isnot perfectly complementary to any version of the target but thathybridizes to all variants during primer annealing. We prefer DNA anchorsequences that form anchor-sequence/target hybrids generally in therange of 15-30 base pairs, as is typical for conventional PCR primers.We demonstrate in the Examples below anchor sequences that are24-nucleotides long, that are DNA, and that are fully complementary tothe target sequence. The multi-part primer does not prime sequences inthe reaction mixture other than its target sequence, that is, theintended target sequence and the unintended, mismatched target sequence.Whereas a conventional primer must be designed to achieve that function,the requirement for an anchor sequence is less strict, because the footsequence aids in discriminating against other sequences that are or maybe present in a sample.

Referring to FIG. 1, the primer includes a foot sequence 106 that iscomplementary to the intended target sequence in the region thatincludes the nucleotide (the SNP nucleotide), or in some cases twonucleotides, that are different from the unintended, mismatched targetsequence. The foot sequence may be perfectly complementary to theintended target sequence, or it may contain one or, in some cases, eventwo nucleotides that are mismatched to both the intended target sequenceand the unintended target sequence. Foot sequence 106 is always morecomplementary to the intended target sequence than to the mismatchedtarget sequence by at least one nucleotide. The foot sequence is copiedduring amplification. Because exponential amplification proceeds rapidlywith high, normal PCR efficiency, the inclusion of non-naturalnucleotides, nucleotide mimics, and non-natural internucleotide linkagesis limited to types and numbers that permit rapid and efficient copyingby DNA polymerase. We prefer that foot sequences be DNA sequences.Because it is desirable that subsequent exponential amplification ofamplicons proceed with high, normal PCR efficiency, the inclusion in thefoot sequence of non-natural nucleotides, nucleotide mimics, andnon-natural internucleotide linkages is limited to types and numbersthat permit efficient copying by DNA polymerase. In preferredembodiments the foot sequence is a DNA sequence that is perfectlycomplementary to the intended target sequence and contains a singlenucleotide that is mismatched to a nucleotide in the unintended targetsequence.

Foot sequence 106 forms a hybrid with the intended target sequence thatis at least 5 nucleotides long, for example, in the range of 5-8 basepairs, preferably in the range of 6-8 base pairs, and more preferablynot longer than 7 nucleotides long, for example, in the range of 6-7base pairs. When the anchor sequence is hybridized to the intendedtarget sequence, there is only one binding site for the foot sequence.As the foot sequence is shortened, the chance is increased that it couldhave another possible binding site, particularly if the foot sequence isshortened to just 5 nucleotides, a matter to be taken into account inprimer design. While, as we demonstrate in the Examples, the mismatchednucleotide versus the unintended target may occur at any nucleotideposition of foot 106, we prefer that the mismatched nucleotide either bethe 3′ terminal nucleotide, as in an ARMS primer (Newton et al. (1989)Nucleic Acids Res. 17:2503-2516; and Ferrie et al. (1992) Am. J. Hum.Genet. 51:251-262) or reside one nucleotide in from the 3′ end of thefoot, which we sometimes refer to as the “3′ penultimate nucleotide.”

Again referring to FIG. 1, the primer includes a bridge sequence 105that is chosen so that it cannot hybridize with the intervening sequence109 during the annealing of the multi-part primer to a target molecule.The bridge sequence or, if it contains a blocking group, the 3′ portionthereof, is copied by DNA polymerase. Because it is desired thatexponential amplification of the amplicons proceed rapidly with high,normal PCR efficiency, the inclusion in the bridge sequence's copiedportion of non-natural nucleotides, nucleotide mimics, and non-naturalinternucleotide linkages is limited to types and numbers that permitrapid and efficient copying by DNA polymerase. Bridge sequences that areDNA are preferred.

The bridge sequence 105 and its opposed intervening sequence 109 in thetarget form a bubble in the primer/intended target hybrid. Thecircumference of the bubble is the length of bridge sequence 105 plusthe length of intervening sequence 109, plus 4 (a pair of nucleotidesfrom the anchor-sequence hybrid and a pair of nucleotides from thefoot-sequence hybrid). The bridge and intervening sequence need not beof equal length: either can be shorter than the other. In certainembodiments the length of the intervening sequence can be zero. Inpreferred embodiments it is at least six nucleotides long. In morepreferred embodiments wherein the sum of the lengths of the bridge andintervening sequences is at least 24 nucleotides, we prefer that theintervening sequence have a length of at least eight nucleotides, morepreferably at least ten nucleotides. The bridge sequence should be atleast six nucleotides long. Certain preferred embodiments have bridgeand intervening sequences that are equal in length. The circumference ofthe bubble may be as short as 16 nucleotides and as long as 52nucleotides, for example 16-52 nucleotides, 20-52 nucleotides, or 28-44nucleotides.

As general considerations for design of multi-part primers, increasingthe circumference of the bubble and shortening the foot increases thedelay in amplification of the intended target. The number of PCR cyclesneeded to synthesize a predetermined detectable number of amplicons in areaction initiated with a particular number of intended target sequences(the threshold cycle, C_(T), for that reaction) can be measured, forinstance, by observing the fluorescence intensity of the intercalatingdye SYBR® Green, whose intensity reflects the number of ampliconspresent during each PCR cycle. This provides a method for measuring thedifference in probability that a DNA polymerase extends multi-partprimer/unintended-target hybrids relative to the probability that theDNA polymerase extends multi-part primer/intended target hybrids. Giventhat amplification proceeds by exponential doubling, a C_(T) differenceof 10 cycles indicates that the probability of extension of a multi-partprimer/unintended-target hybrid is 1,000 times lower than theprobability of extension of the multi-part primer/intended-targethybrid; a C_(T) difference of 13.3 cycles indicates that the probabilityis 10,000 times lower; a C_(T) difference of about 16.6 cycles indicatesthat the probability is 100,000 times lower; and a C_(T) difference of20 cycles indicates that the probability is one-million times lower.

In an assay according to this invention utilizing multi-part primers,the difference between the higher threshold cycle observed formismatched target sequences and the lower threshold cycle observed forthe same number, for example 10⁶ copies, of intended target sequences,as reflected in the ΔC_(T) from measurements of fluorescence intensityat each PCR cycle achieved by adding SYBR® Green dye to the reactionmixture, should be at least 10 cycles, preferably at least 12 cycles,more preferably at least 14 cycles, even more preferably at least 17cycles, even more preferably at least 18 cycles, and most preferably 20cycles or more. In amplification reactions wherein a multi-part primeraccording to this invention replaces a well-designed conventional PCRprimer, there is a delay (ΔC_(T)) in the threshold cycle achieved usingthe intended target sequence. The amount of delay depends on how wellthe compared conventional primer is designed, but typically, comparingto a conventional primer consisting of just the anchor sequence of themulti-part primer, the delay is at least two amplification cycles, oftenat least three cycles, and sometimes at least eight cycles, or even tencycles.

Preferred embodiments of methods according to this invention includedetecting product resulting from amplification of the rare targetsequence. Detection of amplified product may be performed separatelyfollowing amplification, for example, by gel electrophoresis. Inpreferred embodiments, detection reagents are included in theamplification reaction mixture, in which case detection may be “realtime,” that is, performed on multiple occasions during the course ofamplification, or “end point,” that is, performed after conclusion ofthe amplification reaction, preferably by homogeneous detection withoutopening the reaction container. Detection reagents include DNA bindingdyes, for example SYBR® Green, dual-labeled fluorescent probes thatsignal production of amplified product, for example, molecular beaconprobes, and a combination of a binding dye and a fluorescent probe thatis stimulated by emission from the dye. In addition, as describedherein, the primers themselves can include fluorescent labels that onlyfluoresce when the primer is incorporated into an amplicon, oralternatively, when the primer binds to a complementary amplicon.

This invention includes reaction mixtures for amplifying at least onetarget sequence. Reaction mixtures include a pair of primers for eachintended target sequence, one primer in each pair being a multi-partprimer as described herein. Reaction mixtures also include reagents foramplifying the targets, including deoxyribonucleoside triphosphates,amplification buffer, and DNA polymerase. Preferred reaction mixturesfor assay methods according to this invention also include detectionreagents, that is, DNA binding dye, hybridization probes (or both), or a5′ functional tail of each multi-part primer. If the starting samplescontain RNA, the amplification reaction mixtures may also includereverse transcriptase and primers for reverse transcription.

This invention also includes products that are kits for performing theamplification reactions and amplification-and-detection reactionsdescribed above for one or more intended target sequences. A kitincludes oligonucleotides and reagents needed to create a reactionmixture according to this invention. A kit for starting samples that areRNA may include reagents for reverse transcription.

The details of one or more embodiments of the invention are set forth inthe description below. Other features, objectives, and advantages of theinvention will be apparent from the description and from the claims.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a schematic representation of a multi-part primer useful inthis invention hybridized to its intended target sequence and hybridizedto a mismatched sequence differing from the intended target sequence byone or more nucleotide substitutions.

FIG. 2 is a schematic representation of the amplification cycle in whicha multi-part primer of this invention is first copied, as well assubsequent copying of the resulting amplicon in the next two cycles.

FIG. 3 is a schematic representation of a multi-part primer according tothis invention showing locations for placement of a blocking group thatterminates copying by a DNA polymerase.

FIG. 4 is a schematic representation of several exemplary optional 5′functional moieties.

FIG. 5 shows the real-time fluorescence results obtained with aconventional linear primer and either 1,000,000 intended targetsequences or 1,000,000 unintended, mismatched target sequences thatdiffer from each other at a single nucleotide located in the middle ofthe sequence to which the primers bind.

FIG. 6 shows the real-time fluorescence results obtained with an ARMSprimer and either 1,000,000 intended target sequences or 1,000,000unintended, mismatched target sequences differing by a singlenucleotide, where the “interrogating nucleotide” in the primer (which iscomplementary to the corresponding nucleotide in the intended targetsequence, but not complementary to the corresponding nucleotide in theunintended target sequence) is the 3′-terminal nucleotide of the primer;and the figure also shows the results obtained with a similar primerwhere the interrogating nucleotide is at the penultimate nucleotide fromthe 3′ end of the primer.

FIG. 7 shows the real-time fluorescence results obtained with amulti-part primer according to this invention in reactions containingeither 1,000,000 molecules of the primer's intended target sequence or1,000,000 molecules of the primer's unintended target sequence (wherethe multi-part primer possessed an interrogating nucleotide at thepenultimate position of the foot sequence).

FIG. 8 shows the real-time fluorescence results obtained with amulti-part primer according to this invention in a series of reactionsthat each contains 1,000,000 unintended target sequences and either: 0;10; 100; 1,000; 10,000; 100,000; or 1,000,000 intended target sequences.

FIG. 9 is a graph showing the inverse linear relationship between thethreshold cycle observed for each reaction shown in FIG. 8 versus thelogarithm of the number of intended targets present in each reaction,and a dotted line in the figure indicates the threshold cycle obtainedfor the reaction that contained 1,000,000 unintended target sequencesand no intended target sequences.

FIG. 10 is a graph showing the results that were obtained with the samedilution series used for the experiment shown in FIG. 8 and FIG. 9,utilizing three otherwise identical multi-part primers whose foot waseither 6, 7, or 8 nucleotides in length (where the interrogatingnucleotide was located at the penultimate position in each footsequence).

FIG. 11 is a graph showing the results that were obtained with the samedilution series used for the experiment shown in FIG. 8, FIG. 9, andFIG. 10, utilizing three multi-part primers whose bridge sequences formbubbles of different circumferences with an identical-length interveningsequence in the target molecules.

FIG. 12 is a series of graphs showing the real-time fluorescence resultsobtained with otherwise identical multi-part primers according to thisinvention and either 1,000,000 intended target sequences or 1,000,000unintended target sequences (differing from the intended target sequenceby a single-nucleotide polymorphism), where the interrogating nucleotidein the foot of the primer (which is complementary to the correspondingnucleotide in the intended target sequence, but not complementary to thecorresponding nucleotide in the unintended target sequence) is locatedat different positions relative to the 3′ end of the primer.

FIG. 13 is a series of graphs showing the real-time fluorescence resultsobtained with multi-part primers according to this invention and either1,000,000 intended target sequences or 1,000,000 unintended targetsequences differing by a single nucleotide, in which the length of thebridge sequence plus the length of the intervening sequence in thetarget molecule is held constant (i.e., the circumference of the bubbleis the same), but where the symmetry of the bubble formed by the bridgesequence and intervening sequence in the target molecule (relativelengths of those sequences) is varied.

FIG. 14 is a graph showing the inverse linear relationship between thethreshold cycle observed and the logarithm of the number of V600E mutanthuman B-raf target sequences in a series of reactions that eachcontained 1,000,000 wild-type human B-raf target sequences, and either:10; 100; 1,000; 10,000; 100,000; or 1,000,000 V600E mutant human B-raftarget sequences. The dotted line indicates the threshold cycle obtainedfor a reaction that contained DNA from 1,000,000 wild-type human B-raftarget sequences and no DNA from V600E mutant human B-raf targetsequences.

FIG. 15 is a graph showing the inverse linear relationship between thethreshold cycle observed and the logarithm of the number of mutanttarget sequences present in a series of reactions that each contained10,000 wild-type target sequences present in genomic DNA isolated fromcultured normal human cells and either: 10; 30; 100; 300; 1,000; 3,000;or 10,000 mutant target sequences present in genomic DNA isolated fromcultured human cancer cells possessing the T790M mutation in the EGFRgene. The dotted line indicates the threshold cycle obtained for areaction that contained 10,000 wild-type target sequences and no DNAfrom cancer cells.

FIG. 16 shows the results of an experiment that is similar to theexperiment whose results were shown in FIG. 9, except that an AppliedBiosystems PRISM 7700 spectrofluorometric thermal cycler was used tocarry out the experiment, instead of a Bio-Rad IQ5 spectrofluorometricthermal cycler.

FIG. 17 shows the real-time fluorescence results obtained, panel A, witha multi-part primer according to this invention in reactions containingeither 1,000,000 molecules of the primer's intended target sequence or1,000,000 molecules of the primer's unintended target sequence (wherethe multi-part primer possessed an interrogating nucleotide at thepenultimate position of the foot sequence), and, panel B, with atruncated version of the primer missing the 3′-penultimate and3′-terminal nucleotides.

FIG. 18 is a schematic representation of two multi-part primersaccording to this invention that may be used in a multiplex reaction fortwo closely related intended target sequences.

FIG. 19 is a schematic representation of two multi-part primers and twomolecular beacon probes that may be used in a multiplex reaction for twoclosely related intended target sequences.

DETAILED DESCRIPTION

This invention is based, at least in part, on a unique design ofmulti-part primers for primer-dependent amplification reactions.Accordingly, this invention discloses the design and characteristics ofmulti-part primers, which exhibit extraordinary selectivity when theyare hybridized to the templates that are present in the original sample.Due to this extraordinary selectivity, we call the multi-part primers ofthis invention “SuperSelective” primers.

Significantly, once synthesis is initiated on mutant templates, theresulting amplicons are exponentially amplified with high efficiency,and the real-time data provide a conventional means of assessing theabundance of the mutant templates present in the original sample. Theexperiments described below demonstrate that SuperSelective primers aresufficiently discriminatory to suppress the synthesis of wild-typesequences to such an extent that as few as 10 molecules of a mutantsequence can be reliably detected in a sample containing 1,000,000molecules of the wild-type sequence, even when the only differencebetween the mutant and the wild-type is a single-nucleotidepolymorphism.

1. PRIMER-DEPENDENT AMPLIFICATION REACTIONS

Primer-dependent amplification reactions useful in methods of thisinvention may be any suitable exponential amplification method,including the polymerase chain reaction (PCR), either symmetric ornon-symmetric, the ligase chain reaction (LCR), the nicking enzymeamplification reaction (NEAR), strand-displacement amplification (SDA),nucleic acid sequence-based amplification (NASBA),transcription-mediated amplification (TMA), and rolling circleamplification (RCA). Preferred methods utilize PCR. In non-symmetric PCRamplification methods, for example asymmetric PCR, one primer, thelimiting primer, is present in a limiting amount so as to be exhaustedprior to completion of amplification, after which linear amplificationoccurs, using the remaining primer, the excess primer. A non-symmetricPCR method useful in this invention is LATE-PCR (see, for example,European Patent EP 1,468,114; and Pierce et al. (2005) Proc. Natl. Acad.Sci. USA 102:8609-8614). If a non-symmetric amplification method isused, the multi-part primer is preferably the excess primer. Preferredmethods also include digital PCR (see, for example, Vogelstein andKinzler (1999) Proc. Natl. Acad. Sci. USA 98:9236-9241), where it isdesirable to detect a large number of amplicons from a single mutanttemplate molecule that is present in reactions that contain abundantwild-type molecules.

If the amplification reaction utilizes an RNA-dependent DNA polymerase(an example being NASBA), the amplification reaction is isothermal. Werefer to repeated rounds of synthesis of amplified product as “cycles”,but they are not thermal cycles. For such amplification the “intendedtarget sequence” and the “unintended target sequence” that are primed bya multi-part primer according to this invention are RNA sequences thatoccur in an original sample and in the amplification reaction mixture,where they are present with the DNA polymerase and the multi-partprimer.

If the amplification reaction utilizes a DNA-dependent DNA polymerase(an example being PCR), an original sample may contain either DNA or RNAtargets. For such amplifications, the “intended target sequence” and the“unintended target sequence” that are primed by a multi-part primeraccording to this invention are DNA sequences that either occur in anoriginal sample or are made by reverse transcribing RNA sequences thatoccur in the original sample. If the multi-part primer is used forreverse transcription, the “intended target sequence” and the“unintended target sequence” are RNA as well as cDNA. If a separate,outside primer is used for reverse transcription, the “intended targetsequence” and the “unintended target sequence” are cDNA. In either case,the “intended target sequence” and the “unintended target sequence” arenucleic acid sequences that are present in the amplification reactionmixture with the DNA polymerase and the multi-part primer.Primer-dependent amplification reactions comprise repeated thermalcycles of primer annealing, primer extension, and strand denaturation(strand melting). Primer annealing may be performed at a temperaturebelow the primer-extension temperature (for example, three-temperaturePCR), or primer annealing and primer extension may be performed at thesame temperature (for example, two-temperature PCR). The overall thermalprofile of the reaction may include repetitions of a particular cycle,or temperatures/times may be varied during one or more cycles. Forexample, once amplification has begun and the priming sequence of amulti-part primer is lengthened, a higher annealing temperatureappropriate for the longer primer might be used to complete theamplification reaction.

Assay methods according to this invention include detection of anamplified target sequence. Methods according to this invention are notlimited to particular detection schemes. Detection may be performedfollowing amplification, as by gel electrophoresis. Alternately,homogeneous detection may be performed in a single tube, well, or otherreaction vessel during (real time) or at the conclusion (end point) ofthe amplification reaction using reagents present during amplification.Alternatively, using a microfluidic device, amplified products can bemoved to a chamber in which they contact one or more detection reagentsor isolating reagents, such as immobilized capture probes. Detectionreagents include double-stranded DNA binding dyes, for example SYBRGreen, and fluorescently or luminescently labeled hybridization probesthat signal upon hybridization, for example molecular beacon probes orResonSense® probes, or probes that are cleaved during amplification, forexample 5′-nuclease (TaqMan®) probes.

2. MULTI-PART PRIMER

As discussed above, methods of this invention include use of amulti-part primer for each rare target sequence. Amplification with amulti-part primer is illustrated in FIG. 2 for primer 103 and intendedtarget sequence 101 (FIG. 1). First, primer 103, shown as a forwardprimer, anneals to target sequence 101 and is extended by a DNApolymerase using strand 101 as a template to produce extension product201. Referring to the middle sketch, in the next amplification cyclestrand 202, which comprises primer 103 and extension product 201,becomes a template for the reverse primer, a conventional primer 203.Reverse primer 203 anneals and is extended by the DNA polymerase usingstrand 202 as a template to produce extension product 204. It will beobserved that extension product 204 includes a sequence perfectlycomplementary to primer 103. Extension product 204 includes such aperfectly complementary sequence irrespective of the sequence of strand101. That is, if primer 103 has been extended in the earlier cycle (topsketch), the resulting strand 202 (middle sketch) always includes theperfect complement of primer 103. In the next amplification cycle (lowersketch), strand 205, which comprises reverse primer 203 and extensionproduct 204, contains the perfect complement of primer 103; and primer103 binds to strand 205 and is extended by a DNA polymerase to produceextension product 206. Thus, FIG. 2 applies to mismatched targetsequence 102, as well as to intended target sequence 101, any time thatthe multi-part primer anneals and is extended to generate amplicon 202.

As indicated in the preceding paragraph, FIG. 2 shows copying of theentirety of primer 103 during extension of reverse primer 203. Thatcreates a long priming region for the next cycle, namely, a sequencecomplementary to anchor sequence 104, bridge sequence 105 and footsequence 106. In certain embodiments it may not be desired to proceedwith the remainder of amplification with a priming region of suchlength. FIG. 3 illustrates the use of multi-part primers that possess ablocking group to shorten the priming region in later cycles. Blockinggroups are well known for stopping extension by a DNA polymerase. Ablocking group may be, for example, hexethylene glycol (HEG).Particularly if bridge sequence 105 is long, it may be desirable toplace a blocking group 108 in bridge sequence 105, as shown in the topsketch of FIG. 3. The priming region in later amplification cycles willconsist of the nucleotides of foot 106 plus nucleotides of bridge 105that are located 3′ of blocking group 108. Alternatively, it may bedesirable to place a blocking group 108A in anchor sequence 104, asshown in the bottom sketch of FIG. 3. In such an embodiment, the primingregion in later cycles of amplification will include the nucleotides offoot 106, the nucleotides of bridge 105, plus nucleotides of anchor 104that are located 3′ of blocking group 108A.

As stated above, a multi-part primer for use in this invention mayinclude a functional moiety, a 5′ tail attached to anchor sequence 104.This invention is not limited as to the function such a group mayperform or as to the structure thereof. Examples of several functionalmoieties are illustrated in FIG. 4. Each drawing shows a multi-partprimer 103 with anchor sequence 104 and a different functional grouplocated at the 5′ end of the anchor sequence. Functional group 401 issimply an oligonucleotide tail that can be used for hybridization to acapture probe or hybridization to a labeled probe. Tail 401, asdepicted, is not complementary to another sequence within primer 103.Because of the presence of blocking group 108B in the primer containingTail 401, DNA polymerase does not copy Tail 401, and Tail 401 is alwayssingle stranded and available to bind to a capture probe or to a labeledprobe, irrespective of whether the complementary amplicons are singlestranded or double stranded. Oligonucleotide 401 may serve as a “zipcode” for the immobilization of the resulting amplicons to a specificposition on an array of capture probes, or to capture probes linked todifferent elements of a distributed array. Another functional moietyincludes biotin group 402 attached to anchor sequence 104 through linker403. Because of the presence of blocking group 108B in the primer, DNApolymerase does not copy linker 403, and linker 403 is always singlestranded. Biotin group 402 enables the amplicons synthesized from theprimer to acquire an additional function. For example, a biotin groupallows amplicons to be strongly captured by streptavidin proteins thatare immobilized through a linking group to a solid surface, such as aparamagnetic bead. Another functional moiety is hairpin oligonucleotide404 having a stem-and-loop structure comprising single-stranded loop 405and double-stranded stem 406 that is labeled with quencher 407(preferably a non-fluorescent quencher such as Dabcyl or Black HoleQuencher 2) and an interacting fluorescent moiety 408 (preferably afluorophore). Extension of reverse primer 203 (FIG. 2) would continuethrough labeled hairpin 404, separating quencher 407 from fluorescentmoiety 408, thereby generating a fluorescent signal. See Nazarenko etal. (1997) Nucleic Acids Res. 25:2516-2521. Inclusion of labeled hairpin404 in primer 103 leads to a fluorescent signal indicative ofamplification. Yet another functional moiety is a molecular beacon probe409 attached to anchor sequence 104 through oligonucleotide sequence 414and blocking group 108B. This functional moiety has the additionalfunction of a Scorpion® primer, that is, enabling the tethered molecularprobe to hybridize to the target strand (both the intended targetsequence and the mismatched target sequence) downstream from primer 103as copy 201 is generated. Molecular beacon probe 409 comprises loop 410and stem 411 covalently attached to which are interacting quencher 412and fluorescent moiety 413, such that hybridization of probe 409 toextension product 201 disrupts stem 411 and generates a fluorescentsignal indicative of amplification. Unlike hairpin 404, hairpin 409 isnot copied, because in this case primer 103 contains blocking group108B. The drawing at the bottom of FIG. 4 depicts a variant of hairpin404 in which the 5′-terminal sequence of the stem 417 of the molecularbeacon is complementary to a portion of bridge sequence 105 and the loopcomprises anchor sequence 104. Consequently, upon hybridization to acomplementary amplicon strand, the rigidity of the resulting hybridseparates interacting quencher 415 from fluorescent moiety 416, therebygenerating a fluorescent signal indicative of amplification.

The multi-part primer does not prime sequences in the reaction mixtureother than its target sequence, that is, the intended target sequenceand the unintended, mismatched target sequence. The 3′ portion of thebridge sequence plus the foot sequence do not together form a sequencethat serves as a primer for such irrelevant sequences.

A multi-part primer useful in methods of this invention functions asfollows, with reference to FIG. 1. In the first round of synthesis, forexample, in the first PCR cycle, which may follow a high-temperaturedenaturation step, anchor sequence 104 hybridizes to the targetsequence, both the intended target 101 and the unintended, mismatchedtarget 102. Bridge sequence 105 does not hybridize to the targetsequence. Foot sequence 106 hybridizes preferentially to intended targetsequence 101, but to some extent it hybridizes also to unintended,mismatched target sequence 102. The hybrids form and separate with somefrequency. Also with some frequency, a DNA polymerase binds to theformed hybrids and initiates extension of the primer. With respect tointended target sequence 101, the combined frequencies of hybridformation and polymerase binding/extension result in inefficient copyingof intended target sequence 101, which we measure as a delay in the PCRthreshold cycle, C_(T), of at least two cycles when comparison is madebetween a PCR amplification and detection assay with SYBR Greendetection using the multi-part primer and 10⁶ copies of intended targetsequence 101 (with or without copies of unintended target sequence 102)and the same assay using a corresponding conventional primer (which issimilar to the anchor sequence in multi-part primers). With respect tounintended, mismatched target sequence 102, the combined frequencies ofhybrid formation and polymerase binding/extension result in extremelyinefficient copying, which we measure as a difference (ΔC_(T)) in suchan assay between the C_(T) with the multi-part primer and 10⁶ copies ofmismatched target sequence 102 and the C_(T) with the multi-part primerand 10⁶ copies of intended target sequence 101 (with or without copiesof unintended target sequence 102). The delay for the intended targetsequence caused by the multi-part primer is at least two PCR cycles, andmay be larger, for example, four cycles or even 5-10 cycles. Thedifference (ΔC_(T)) between the unintended, mismatched target and theintended target is at least ten PCR cycles, preferably more. Theintended target sequence will be copied as amplification proceedsthrough additional cycles, and, eventually, so will the mismatchedtarget. Synthesized copies from both targets will contain the multi-partprimer and so will be identical.

After a multi-part primer initiates the synthesis of an amplicon on atarget nucleic acid molecule that was present in the sample to be testedprior to amplification, whether that initiation occurs in the firstcycle or in a later cycle, the resulting amplicon is then exponentiallyamplified in subsequent cycles rapidly with normal, high efficiency,with the multi-part primer acting as a conventional primer with respectto the amplicons. For example, for the copying of amplicons, themulti-part primer functions in the same manner as a conventional PCRprimer that is 20-50 nucleotides long. This means that more than thefoot acts as a primer once amplification has begun. One possibility isthat the entirety (or at least the entirety except for a functionalmoiety located 5′ to a blocking group, such as 401, 403, and 409) iscopied and acts as a primer for the copying of amplicons.

In those embodiments that possess a blocking group in the multi-partprimer, the purpose of the blocking group is to prevent copying of someportion of the primer's 5′ end. Blocking groups are familiar to personsskilled in the art. A blocking group may be, for example, hexethyleneglycol or an abasic nucleotide that lacks a nitrogenous base. A blockinggroup may be placed to the 5′ end of anchor sequence 104 to preventcopying of a functional moiety, such as the placement of blocking group108B with respect to functional moieties 401, 403, or 409; or it may beplaced at any location within anchor sequence 104, such as the placementof blocking group 108A; or it may be placed within bridge sequence 105,such as the placement of blocking group 108; just so long as theshortened sequence that is copied is sufficiently long to act as anefficient primer when the template molecules are amplicons. Toillustrate, suppose that a multi-part primer has a foot sequence sixnucleotides long and that one wishes that 35 nucleotides be copied. Ifbridge sequence 105 is twenty-four nucleotides long, five nucleotides ofthe anchor sequence 104 must be downstream (that is, 3′) of a blockinggroup to achieve the desired primer length.

3. NOMENCLATURES

In the Examples disclosed below, two nomenclatures are used to refer toa number of multi-part primers of this invention.

In one nomenclature, a multi-part primer is referred to in such a formatas, e.g., a “24-14-5:1:1” primer, referring to an anchor sequence thatis 24 nucleotides long, a bridge sequence that is 14 nucleotides long,and a foot sequence that is seven nucleotides long (comprising, from the5′ end of the foot, five nucleotides complementary to both the mutant(MUT) and wild type (WT) targets, one interrogating nucleotide that isnot complementary to the corresponding nucleotide in the WT target, butthat is complementary to the corresponding nucleotide in the MUT target,and, finally, one nucleotide complementary to both targets. Because theinterrogating nucleotide is located one nucleotide inboard of the 3′ endof the primer, we refer to this nucleotide as being located at the“3′-penultimate position.”

Comparing the bridge sequence to the region of the target sequence lyingbetween the binding sequence of the anchor and the binding sequence ofthe foot, which we call the “intervening sequence,” one can see that theintervening sequence in some of the Examples below is fourteennucleotides long, the same length as the bridge sequence while in others(such as Example 8) the intervening sequence and the bridge sequencehave different lengths. To specify the length of the interveningsequence, a second nomenclature is sometimes used. In that case, a“24-18/10-5:1:1” multi-part primer indicates that its 5′-anchor sequenceis 24-nucleotides long, its bridge sequence is 18-nucleotides long andoccurs opposite an intervening sequence in the template that is10-nucleotides long, and its 3′-foot sequence is 7-nucleotides long andconsists of a 5′ segment that is fully complementary to both the mutantand to the wild-type templates, followed by an interrogating nucleotidethat is only complementary to the corresponding nucleotide in the mutanttemplate, followed by a 3′ nucleotide that is complementary to thecorresponding nucleotide in both the mutant and the wild-type templates.

The sequence of the bridge sequence is chosen so that it is notcomplementary to the intervening sequence, in order to prevent thehybridization of the bridge sequence to the intervening sequence duringprimer annealing. Instead of annealing to each other, the bridgesequence and the intervening sequence form a single-stranded “bubble”when both the anchor sequence and the foot sequence are hybridized tothe template. We sometimes refer to the combination of a bridge sequenceand an intervening sequence as a bubble. For example, the designation24-14/14-5:1:1 may be said to have a “14/14 bubble.”

The “circumference of the bubble” is defined as the sum of the number ofnucleotides in the bridge sequence plus the number of nucleotides in theintervening sequence plus the anchor sequence's 3′ nucleotide and itscomplement plus the foot sequence's 5′-terminal nucleotide and itscomplement. Consequently, the circumference of the bubble formed by thebinding of a 24-14/14-5:1:1 multi-part primer (a 14/14 bubble) to thetemplate molecules is 14+14+2+2, which equals 32 nucleotides in length.The listing below lists some of the primers used in the Examples below,utilizing this second format.

Exemplary Primers Utilized in PCR Assays

SEQ ID Primer Sequence (5′ to 3′) NO: EGFR L858R Conventional ForwardCTGGTGAAAACACCGCAGCATGTC 27 Conventional ReverseGCATGGTATTCTTTCTCTTCCGCA 3 24-14/14-5:1:1CTGGTGAAAACACCGCAGCATGTCGCACGAGTGAGCCCTGGGC G G 6 24-14/14-4:1:1TGGTGAAAACACCGCAGCATGTCACACGAGTGAGCCCCGGGC G G 7 24-14/14-5:1:1CTGGTGAAAACACCGCAGCATGTCGCACGAGTGAGCCCTGGGC G G 6 24-14/14-6:1:1ACTGGTGAAAACACCGCAGCATGTTGGAGCTGTGAGCCTTGGGC G G 8 24-14/14-6:1:0ACTGGTGAAAACACCGCAGCATGTTGCACGAGTGAGCCTTGGGC G 11 24-14/14-5:1:1CTGGTGAAAACACCGCAGCATGTCGCACGAGTGAGCCCTGGGC G G 6 24-14/14-4:1:2TGGTGAAAACACCGCAGCATGTCACACGAGTGAGCCACGGGC G GG 12 24-14/14-3:1:3GGTGAAAACACCGCAGCATGTCAAACGAGTGAGCCACAGGC G GGC 13 24-14/14-2:1:4GTGAAAACACCGCAGCATGTCAAGGAAGTGAGCCACAAGC G GGCC 14 24-14/14-1:1:5TGAAAACACCGCAGCATGTCAAGACAGACTGACCCAAAC G GGCCA 15 24-10/10-5:1:1TGAAAACACCGCAGCATGTCAAGACACTCAGCCCTGGGC G G 10 24-14/14-5:1:1CTGGTGAAAACACCGCAGCATGTCGCACGAGTGAGCCCTGGGC G G 6 24-18/18-5:1:1CGTACTGGTGAAAACACCGCAGCACTGACGACAAGTGAGCCCTGGGC G G 9 24-18/10-5:1:1TGAAAACACCGCAGCATGTCAAGACACACGACAAGTGAGCCCTGGGC G G 16 24-16/12-5:1:1GGTGAAAACACCGCAGCATGTCAATCCAACAAGTGAGCCCTGGGC G G 17 24-14/14-5:1:1CTGGTGAAAACACCGCAGCATGTCGCACGAGTGAGCCCTGGGC G G 6 24-12/16-5:1:1TACTGGTGAAAACACCGCAGCATGGACGACGAGCCCTGGGC G G 18 24-10/18-5:1:1CGTACTGGTGAAAACACCGCAGCACTGACGGCCCTGGGC G G 19 B-raf V600E24-14/14-5:1:1 AGACAACTGTTCAAACTGATGGGAAAACACAATCATCTATTTC T C 20Conventional Reverse ATAGGTGATTTTGGTCTAGC 22

The bridge sequence within each SuperSelective primer is underlined, andthe interrogating nucleotide in its foot sequence is represented by anunderlined bold letter. The primers are arranged into groups thatreflect their use in comparative experiments.

4. USES

This invention is not limited to particular intended targets, particularamplification methods, or particular instruments. For comparativepurposes we present in Examples 1-8 several series of experiments thatutilize the same intended target, EGFR mutation L858R, a homogeneous PCRassay starting with plasmid DNA, utilizing SYBR® Green detection, andusing the same thermal cycler, a Bio-Rad IQ5 spectrofluorometric thermalcycler. We have performed other assays that gave results consistent withthose reported in the Examples. Such assays have utilized other intendedtargets, including human EGFR mutant T790M and human B-raf mutant V600E;have utilized genomic DNA; have included detection with molecular beaconprobes; have utilized different PCR parameters; and have utilized adifferent instrument, the ABI PRISM 7700 spectrofluorometric thermalcycler.

Example 1 is a control assay in which a conventional PCR forward primer21-nucleotides long was used to amplify a perfectly matched intendedtarget sequence and also to amplify an unintended, mismatched targetsequence differing by a single-nucleotide polymorphism that is locatednear the middle of sequence to which the primer binds (here, as in otherExamples, a conventional PCR reverse primer was used as well).Homogeneous detection of double-stranded amplification products (ordouble-stranded “amplicons”) was enabled by the inclusion of SYBR Green®in the initial amplification reaction mixture, which binds todouble-stranded amplicons is such a manner as to significantly increasetheir fluorescence. Consequently, the intensity of the SYBR Green®fluorescence measured at the end of the chain elongation stage of eachPCR amplification cycle provides an accurate indication of the number ofamplicons present. Real-time kinetic fluorescence curves (fluorescenceintensity versus amplification cycle number) presented in FIG. 5 showthat the amplifications produced sufficient double-stranded product, onthe order of 10¹² amplicons, to give a detectable signal abovebackground (the threshold cycle, abbreviated “C_(T)”) at the point whereroughly 20 PCR cycles had been carried out, which is typical for a PCRassay starting with 10⁶ templates. FIG. 5 also shows that the forwardprimer had little selectivity in favor of the intended target over theunintended, mismatched target, that is, there was no significant delayin the threshold cycle (C_(T)) when starting with the mismatched target.Thermodynamically, there is little difference in the stability of theperfectly complementary hybrids compared to the stability of themismatched hybrids (resulting in virtually no observable delay in theappearance of amplicons made from the slightly less probable-to-formmismatched primer-target hybrids).

Example 2 describes two additional controls, wherein the substitutednucleotide in the mismatched target was placed first at the 3′ terminalnucleotide of the conventional forward primer, the well-known ARMStechnique, and then at one nucleotide inboard from the 3′ terminalnucleotide of the conventional forward primer. We sometimes refer to thelocation of the nucleotide within a primer sequence that will beopposite the nucleotide in the target where a single-nucleotidepolymorphism can be present or absent as the “interrogating nucleotide.”Real-time kinetic curves for these controls are presented in FIG. 6,where it can be seen that, with the intended target, the C_(T) remainedin the vicinity of 20 cycles, indicating that the amplification reactionwas just as efficient for the intended target as the amplificationreported in Example 1. However, with the mismatched target, the C_(T)was delayed by several cycles. In the case of the primer with theinterrogating nucleotide at the 3′-terminus of the foot sequence, FIG.6A, the delay (11 cycles) was roughly 10 cycles, which indicates aselectivity in favor of the perfectly matched intended target of athousand fold (2¹⁰ is 1,024). In the case of the interrogatingnucleotide being at the penultimate position from the 3′ end of the footsequence, the C_(T) was somewhat less, about 8 cycles. ComparingExamples 1 and 2, one sees that the efficiency of amplification of theintended target is not reduced by placing the interrogating nucleotideat or near the 3′ end of the primer, but selectivity for the intendedtarget over the unintended target differing by a single nucleotide isimproved. We understand that selectivity is limited because, due toketo-enol tautomerism, some base pairing of the mismatched interrogatingnucleotide with the non-complementary nucleotide in the target sequenceoccasionally occurs, and therefore some undesirable extension does takeplace, so the probability of generating an amplicon is the product ofthe probability of a hybrid being formed times the probability that theresulting hybrid forms a structure that can be extended.

Example 3 shows the same experiment with a multi-part primer accordingto this invention. We describe the primer used here as 24-14-5:1:1. Thefirst number, 24, is the nucleotide length of the anchor sequence. Thesecond number, 14, is the nucleotide length of the bridge sequence (andin this experiment, as in the other experiments that are describedherein, except where we explicitly indicate otherwise, the interveningsequence in the target is the same length as the bridge sequence). Thelast three numbers, 5:1:1, describe the foot sequence, giving the numberof nucleotides that are 5′ of the interrogating nucleotide(s), then thenumber of interrogating nucleotides (which is 1 for all of theexperiments described herein), and finally the number of nucleotidesthat are 3′ of the interrogating nucleotide(s). Thus, in this case, thefoot was seven nucleotides long with a penultimate interrogatingnucleotide. The results of these real-time assays, utilizing theintensity of SYBR Green® fluorescence to measure the number of ampliconspresent after the completion of each thermal cycle (determined at theend of the chain elongation stage of each cycle) are presented in FIG.7. Comparing FIGS. 5 and 7, one sees that the C_(T) with the intended,perfectly matched target is delayed, in this case by about 3 cycles. Onealso sees that the C_(T) with the unintended target (containing asingle-nucleotide polymorphism that is not complementary to theinterrogating nucleotide in the foot) is even more delayed, giving aΔC_(T) of about 19 cycles between the intended target sequence and theunintended target sequence, which is approximately a 500,000-folddifference in selectivity (2¹⁹ is 524,288).

While not wishing to be bound by any theory, we believe the following tobe true:

A. Even though the foot sequence is tethered to the template by theanchor hybrid, the foot is so small, and it is separated from the anchorhybrid by such a large bubble (comprising the bridge sequence of theprimer and the intervening sequence in the template), and the annealingtemperature is so high for a short foot sequence, that at any givenmoment (under the equilibrium conditions of the annealing stages of thePCR assay), only a very small portion of the template molecules that arepresent in the sample being tested are hybridized to the foot at anygiven moment.

B. Moreover, the hybrids that do form between the foot and the targetare relatively weak, so the mean time during which they persist is veryshort (perhaps a hundred microseconds).

C. As a consequence of both the reduced probability of a hybrid existingat any given moment, and the reduced mean persistence times of theresulting weak hybrids, there is an extremely low probability of astable (extendable) complex being formed between a hybrid (even aperfectly complementary hybrid) and a DNA polymerase molecule.

D. This is seen in PCR assays carried out with preferred multi-partprimer designs as an approximately 10-cycle delay in the appearance ofthe amplicons made from perfectly complementary (“mutant”) targets (thatis, instead of a C_(T) of about 20, as occurs when conventional linearprimers are utilized with 10⁶ perfectly complementary targets), the Ctis about 30. An increase of 10 thermal cycles in the C_(T) valueindicates that the probability of forming a stable complex between a DNApolymerase molecule and a perfectly complementary foot hybrid is 1/1,000less probable than when a conventional linear primer is utilized underthe same reaction conditions.

E. Under these same PCR conditions, utilizing the same preferredmulti-part primer design, the C_(T) value obtained with mismatched(“wild-type”) targets occurs almost 20 cycles later than the C_(T) valuethat occurs with a perfectly complementary target. There is thus anapproximately 30-cycle delay in the appearance of amplicons from thesemismatched targets compared to the C_(T) value that would have occurredunder the same conditions had a conventional linear primer been used inplace of the multi-part primer. Thus, the probability of forming astable complex between a DNA polymerase molecule and a hybrid containinga foot sequence bound to a mismatched foot target sequence is immenselylower. This 30-cycle increase in the C_(T) value indicates that theprobability of forming a stable complex between a DNA polymerasemolecule and a mismatched foot hybrid is 1/1,000,000,000 less probablethan when a conventional linear primer is utilized under the samereaction conditions.

F. This dramatically lower probability of forming extendable complexesbetween an unintended target sequence and a DNA polymerase molecule isthe product of the following discriminatory elements: (i) the lowerstability of the mismatched hybrid (compared to the stability of theperfectly complementary hybrid) markedly decreases the fraction ofmismatched hybrids present at any given moment (compared to the fractionof perfectly complementary hybrids that can be present at any givenmoment); and (ii) the lower stability of the mismatched hybrids resultsin a shorter mean persistence time for the hybrids, thereby markedlydecreasing the ability of a DNA polymerase molecule (subject to constantBrownian motion) to find a hybrid with which to form a stabilizedcomplex.

Example 4 shows that with the assay of Example 3, one can readilydistinguish the different results obtained with a sample containing only10⁶ copies of the unintended target sequence and a sample containing tenor more copies of the intended target sequence in the presence of 10⁶copies of the unintended target sequence. The real-time PCR resultsobtained for a dilution series (10⁶, 10⁵, 10⁴, 10³, 10², 10¹ copies ofthe intended target sequence in a reaction mixture containing 10⁶ copiesof the unintended target sequence) are presented in FIG. 8, and theC_(T)'s determined for those results are presented in FIG. 9, where theyare plotted against the logarithm of the starting copy number of theintended target sequence. Referring to those figures, one sees that theC_(T) of SYBR Green® fluorescence is delayed by approximately 10 cyclesfor every thousand-fold decrease in the concentration of the intendedtarget, and that a sample with 10 copies of the intended target sequenceplus 10⁶ copies of the unintended target sequence is distinguished froma sample with no intended target sequence and 10⁶ copies of theunintended target sequence; that is, detection of one mutant sequence ina population of 100,000 copies of the corresponding wild-type sequenceis enabled. Further, the assay is quantitative, with the threshold cyclecorresponding to the logarithm of the number of mutant copies in thestarting reaction mixture.

These results confirm the following aspects of the use of selectiveprimers according to this invention:

A. Once a multi-part primer forms a hybrid that binds to a DNApolymerase during an annealing stage of a PCR assay, that stabilizedhybrid is extended during the elongation stages of the PCR assay, andthe resulting amplicons are then amplified with high efficiency (just asthough the reaction was carried out with classical linear primers). Thiscan be seen by the fact that a reduction in the number of mutanttemplates originally present in a sample by a factor of 1,000 results ina delay in the appearance of a significant number of amplicons byapproximately 10 thermal cycles (e.g., in the experiment whose resultsare shown in FIG. 8 and FIG. 9, the C_(T) value of a sample possessing100,000 mutant templates was approximately 27 and the C_(T) value of asample possessing 100 mutant templates was approximately 37). If thenumber of amplicons present efficiently doubles every thermal cycle,then after ten cycles there should be 1,024 times as many amplicons(i.e., 2¹⁰). These results confirm that the amplicons generated from themutant templates present in the sample being tested are then amplifiedefficiently.

B. Efficient amplification of the amplicons occurs because once amulti-part primer is incorporated into the 5′ end of a product amplicon(the “plus” amplicon strand), the complementary amplicon generated inthe next cycle of synthesis (the “minus” amplicon strand) possesses asequence at its 3′ end that is perfectly complementary to the entiresequence of the multi-part primer. Consequently, with respect toamplicons (as opposed to the original template molecules), themulti-part primers behave as though they were classical linear primersfor the further amplification of the amplicons.

C. The extraordinarily selective generation of amplicons from theperfectly complementary mutant templates present in the sample beingtested (compared to the generation of amplicons from the mismatchedwild-type templates present in the sample being tested), combined withthe efficient amplification of the amplicons by the primers once theamplicons are synthesized, enables the resulting real-time data to beused to quantitatively measure the number of mutant template moleculesthat were present in the sample being tested.

There is an inverse linear relationship (in exponential amplificationreactions such as PCR assays) between the logarithm of the number oftarget molecules present in a sample being tested and the number ofthermal cycles that it takes to synthesize a predetermined number ofamplicons, as reflected in the C_(T) values obtained from samplescontaining different numbers of mutant template molecules. See Kramer &Lizardi (1989) Nature 339:401-402. The linearity of a plot of C_(T)versus the logarithm of the number of intended (mutant) templatemolecules present in each sample being tested, as for example in theexperiment whose results are shown in FIG. 9, indicates that there areno significant amplicons being generated from the wild-type templates(even though 1,000,000 wild-type template molecules were present in eachsample). Had there been significant numbers of amplicons generated fromthe wild-type templates, the C_(T) values for samples containing only afew mutant template would have been lower (that is, the results wouldnot have formed a straight line, because the appearance of unwantedamplicons synthesized from the abundant unintended target moleculeswould obscure the appearance of amplicons from very rare intended targetmolecules).

As reported in Example 5, we investigated the effect of the length ofthe foot of a multi-part primer on the amplification reaction using theassay of Example 4 with a series of three probes: 24-14-4:1:1,24-14-5:1:1 and 24-14-6:1:1. The length of the anchor sequence wasmaintained at 24 nucleotides. The length of the bridge sequence wasmaintained at 14 nucleotides, the same single-nucleotide differencebetween the target sequences was maintained, and the location of theinterrogating nucleotide was maintained at the penultimate position fromthe 3′ terminus of the foot. The length of the foot sequence was variedfrom 6 nucleotides to 7 nucleotides to 8 nucleotides by changing thenumber of nucleotides 5′ of the location of the interrogating nucleotidefrom 4 to 5 to 6. The C_(T) values that were obtained are summarized inTable 1 and plotted in FIG. 10 against the logarithm of the startingcopy number of the intended target sequence. Straight lines 1001 (footlength 6), 1002 (foot length 7) and 1003 (foot length 8) are fitted tothe data. It can be seen that all three primers provided quantitativeresults, as reported above for FIG. 9. It can also be seen that fittedlines 1001, 1002 and 1003 are close to parallel, indicating the samequantitative relationship between C_(T) and the logarithm of thestarting copy number for all three foot lengths. FIG. 10 also shows thatshortening the length of the foot delays the C_(T), but as seen in FIG.10, shortening the length of the foot also gives a better straight-linefit of the data from 10⁶ to 10¹ copies of the intended target sequence(that is, the shorter the foot length, the less likely it is thatamplicons synthesized from abundant unintended target molecules in asample being tested will obscure the amplicons synthesized from rareintended target molecules that are present in the same sample).

As reported in Example 6, we also investigated the effect onamplification of the circumference of the bubble formed by the bridgesequence of a multi-part primer and the intervening sequence of theintended and unintended target sequences, using the assay of Example 4with a series of three primers: 24-10-5:1:1, 24-14-5:1:1, and24-18-5:1:1. We maintained the length of the anchor sequence at 24nucleotides; we maintained the foot sequence at 5:1:1; and we varied thelength of the bridge sequence from 10 to 14 to 18 nucleotides, and chosethe sequence of the anchor for each multi-part primer so that theintervening sequence in the target would be the same length as thebridge in that primer. Consequently, the circumference of the bubble(expressed in nucleotides) formed by each of the three primers whentheir foot sequence was hybridized to a target (including the fournucleotides contributed by the anchor hybrid and the foot hybrid) were24, 32, and 40, respectively. The C_(T) values obtained are summarizedin Table 2 and plotted in FIG. 11 against the logarithm of the startingcopy number of the intended target sequence. Straight lines 1101 (bubblecircumference 24), 1102 (bubble circumference 32) and 1103 (bubblecircumference 40) are fitted to the data. It can be seen that all threeprimers provided quantitative results, as reported above for FIG. 9 andFIG. 10. It can also be seen that fitted lines 1101, 1102 and 1103 areclose to parallel, indicating the same quantitative relationship betweenC_(T) and the logarithm of starting copy number for all three bubblecircumferences. FIG. 11 also shows that increasing the circumference ofthe bubble delays the C_(T), but as seen in FIG. 11, increasing thebubble circumference gives a better straight-line fit of the data from10⁶ to 10¹ copies of the intended target sequence (that is, the biggerthe bubble, the less likely it is that amplicons synthesized fromabundant unintended target molecules in a sample being tested willobscure the amplicons synthesized from rare intended target moleculesthat are present in the same sample).

These experimental observations demonstrate that shorter foot lengthsand/or larger bubbles cause hybrid formation to be considerably lesslikely, and shorter foot lengths and/or larger bubbles result inincreased selectivity against mismatched wild-type templates, which isevidenced by the enhanced linearity of plots of C_(T) versus thelogarithm of the number of intended target molecules. In order to gainan understanding of why this is so, we examined the thermodynamics offormation of a foot hybrid under the equilibrium conditions that existduring the annealing stages of PCR assays. Here is our understanding:

A. There is a very high concentration of multi-part primers present inour PCR assays (as there needs to be sufficient multi-part primersavailable to be incorporated into the approximately 10¹³ amplicons thatcan be synthesized in each reaction). Consequently, virtually everytemplate molecule is rapidly bound to the anchor sequence of amulti-part primer under the equilibrium conditions that exist at theannealing stages of these PCR assays. Moreover, because the anchorsequence is long (for example, 24 nucleotides), the bond between theanchor sequence and the template molecules is very strong and persists,on average, for a long time (measured, perhaps, in minutes). Atequilibrium, in a very small portion of these anchored complexes, theshort foot sequence is also hybridized to the template molecule. At anygiven instant, the concentration of anchored complexes whose footsequence is not hybridized is “[A]”, and the concentration of anchoredcomplexes whose foot sequence is hybridized is “[B]”. The classicalequilibrium constant (“k”) that describes the interrelationship thesetwo states is:

k=[B]/[A]  Equation 1

Thermodynamically, the probability of forming a hybrid at equilibriumdepends on both hybrid strength (enthalpy) and on the physicalrelationship that determines the probability that the two sequences willbe able to interact to form a hybrid (entropy). The equilibrium constantcan be determined from the change in enthalpy that occurs uponconversion of an anchored complex whose foot sequence is not hybridizedto a foot sequence that is hybridized (ΔH) and from the change inentropy that occurs upon conversion of an anchored complex whose footsequence is not hybridized to a foot sequence that is hybridized (ΔS),according to the following classical formula:

(ΔH−TΔS)=−RT ln(k)  Equation 2

where R is the thermodynamic gas constant, T is the temperatureexpressed in degrees Kelvin, and ln(k) is the natural logarithm of theequilibrium constant. Rearranging this equation to obtain an expressionfor k:

k=e ^(−(ΔH−TΔS)/RT)  Equation 3

where e=2.71828. For the very same reaction, the fraction of complexesthat possess a hybridized foot sequence (Θ) is described by thefollowing equation: Θ=[B]/([A]+[B]). However, as [B] becomes very small(as is the case for reactions employing multi-part primers), Θapproaches 0, and the equation for Θ can be expressed as follows:

Θ≈[B]/[A]  Equation 4

Since the expression for Θ in Equation 4 is virtually identical to theexpression for k in Equation 1, we can substitute Θ for k in Equation 3,to obtain an equation that relates the very low abundance ofprimer-template complexes that possess a hybridized foot (Θ) to theclassical thermodynamic parameters, ΔH and ΔS, as follows:

Θ=e ^(−(ΔH−TΔS)/RT)  Equation 5

For nucleic acid hybridization reactions that occur under PCRconditions, the quantity (ΔH−TΔS) is a positive value, so e is raised toa negative number, giving a fractional value for Θ. The smaller thevalue of (ΔH−TΔS), the smaller is the fraction Θ. Moreover, during theannealing stages of a PCR reaction, T is constant. Therefore, tounderstand how Θ is altered as a consequence of alterations in thedesign of multi-part primers, we need only consider the magnitude of thevalues of ΔH and ΔS for each primer design, in order to understand theeffect of that design when the multi-part primers are hybridized tointended targets compared to when they are hybridized to unintendedtargets.

B. Entropy is a measure of the number of conformationally distinctstates that a molecular complex can form. Therefore, when the foot of ananchored complex hybridizes to its target, the number of topologicallydistinct states that the complex can form goes from a high number to alow number. Therefore, the change in entropy (ΔS) upon forming a foothybrid has a negative value.

C. Enthalpy is a measure of the stability of a molecular complex,expressed in terms of the amount of energy present in the solutioncontaining the complex. Since high temperatures are required todissociate a nucleic acid hybrid, heat energy is added when the complexis broken apart and heat energy is released upon formation of thecomplex. Therefore, the change in enthalpy (ΔH) upon formation of a foothybrid also has a negative value.

D. The fraction of complexes that possess a hybridized foot sequence(Θ), when multi-part primers are used in PCR assays, is well describedby Equation 5. In the experiments described above, in which the lengthof the foot was varied or the circumference of the bubble was varied,the only variables are ΔH and ΔS. For the formation of foot hybrids, ΔHand ΔS are negative, and the quantity (ΔH−TΔS), which is known as theGibbs free energy (ΔG), is positive. Consequently, the quantity TΔS ismore negative than AH. In terms of calculating the fraction of complexesthat possess a hybridized foot sequence (Θ), the smaller the negativemagnitude of ΔH, the smaller will be Θ. Similarly, the greater thenegative magnitude of ΔS, the smaller will be Θ.

E. In order to determine the effect of different foot lengths on thefraction of complexes that possess a foot hybrid (Θ), it is necessary torealize that, all else being equal, ΔH is less negative the shorter isthe length of the foot hybrid. Consequently, the shorter the length ofthe foot hybrid, the lower is the proportion, at any given moment, ofthe primer-target complexes that possess foot hybrids.

F. Similarly, in order to determine the effect of different bubblecircumferences on the fraction of complexes that possess a foot hybrid(Θ), it is necessary to realize that, all else being equal, ΔS is morenegative the greater the circumference of the bubble. Consequently, thegreater the circumference of the bubble, the lower is the proportion, atany given moment, of the primer-target complexes that possess foothybrids.

G. Given these realizations, now let's look at how the design of thefoot sequences in multi-part primers contributes to the discriminationbetween perfectly complementary target sequences (intended targetsequences) and mismatched target sequences (unintended targetsequences). For example, the multi-part primers used for the experimentwhose results are shown in FIG. 8 and FIG. 9 possessed feet of differentlengths (“6:1:1” or “5:1:1” or “4:1:1”). These designations indicatethat the overall length of each foot was either 8 nucleotides, 7nucleotides, or 6 nucleotides, respectively, with the interrogatingnucleotide (that is either complementary to the corresponding nucleotidein the intended target sequence or not complementary to thecorresponding nucleotide in the unintended) being located at thepenultimate position from the 3′ end of the primer.

H. The reason that we locate the key nucleotide at the penultimateposition is that we believe that when the penultimate base pair cannotform (due to a mismatch) that the terminal base pair also cannot form(even though the 3′ nucleotide of the foot is complementary to thecorresponding nucleotide in the target), because an isolated base pairis extremely unlikely to be stable at the annealing temperature of a PCRassay (approximately 60° C.). Thus, for a given foot sequence, amismatched hybrid will be two base pairs shorter than a perfectlycomplementary hybrid.

I. Here is what this means (conceptually): In order to illustrate thepoint, assume that the temperature (T)=1, and assume that the gasconstant (R)=1, because they are constants. Imagine that the ΔH valuefor the formation of a perfectly complementary hybrid with a 6:1:1 footis −16 and that the ΔH value for the formation of the shorter mismatchedhybrid with a 6:1:1 foot is −12. Let's also imagine that the ΔS valuefor both of these hybrids, which is determined by the circumference ofthe bubble, is −20. Consequently, the ΔG value for the perfectlycomplementary hybrid is 4 (calculated as 20-16), and the ΔG value forthe mismatched hybrid is 8 (calculated as 20-12). Plugging these valuesinto equation 5, the conceptual value of Θ for the hybrid formed with anintended target (Θ_(m)) equals e⁻⁴, which has the value 0.0183. Bycomparison, the conceptual value of Θ for the hybrid formed withunintended target (Θ_(w)) equals e⁻⁸, which has the value 0.000335.There is thus, in this conceptual example, the abundance of perfectlycomplementary hybrids is 54.6 times greater than the abundance ofmismatched hybrids. Although this calculation illustrates that the useof a multi-part primer according to this invention results in a muchlower probability of a foot hybrid formed with an unintended targetbeing present (at any given moment) compared to the probability of afoot hybrid formed with intended target being present (at any givenmoment), and although this difference certainly results in a greaterdelay in the C_(T) for amplicons synthesized from the unintended targetscompared to the C_(T) for amplicons synthesized from the intendedtargets, the actual values of Θ_(m) and Θ_(w) will be different fromthis conceptual example.

J. Now let's do the same conceptual calculation for a multi-part primerpossessing a 5:1:1 foot. In this case, the ΔH value for the formation ofa perfectly complementary hybrid with a 5:1:1 foot is −14 and the ΔHvalue for the formation of a mismatched hybrid with a 4:1:1 foot is −10;and the resulting ΔG values (for the same size bubble, for which ΔS=−20)are as follows: the ΔG value for the perfectly complementary hybrid is 6(calculated as 20−14=6), and the ΔG value for the mismatched hybrid is10 (calculated as 20−10). Plugging these values into equation 5, theconceptual value of Θ for the hybrid formed with an intended target(Θ_(m)) equals e⁻⁶, which has the value 0.00248. By comparison, theconceptual value of Θ for the hybrid formed with unintended target(Θ_(w)) equals e⁻¹⁰, which has the value 0.0000454. Surprisingly, inthis conceptual example, the abundance of perfectly complementaryhybrids is also 54.6 times greater than the abundance of mismatchedhybrids.

K. Now let's do the same conceptual calculation for a multi-part primerpossessing a 4:1:1 foot. In this case, the ΔH value for the formation ofa perfectly complementary hybrid with a 4:1:1 foot is −12 and the ΔHvalue for the formation of a mismatched hybrid with a 4:1:1 foot is −8;and the resulting ΔG values (for the same size bubble, for which ΔS=−20)are as follows: the ΔG value for the perfectly complementary hybrid is 8(calculated as 20−12=8), and the ΔG value for the mismatched hybrid is12 (calculated as 20−8). Plugging these values into equation 5, theconceptual value of Θ for the hybrid formed with an intended target(Θ_(m)) equals e⁻⁸, which has the value 0.000335. By comparison, theconceptual value of Θ for the hybrid formed with unintended target(Θ_(w)) equals e⁻¹², which has the value 0.00000614. And even moresurprisingly, in this conceptual example, the abundance of perfectlycomplementary hybrids is also 54.6 times greater than the abundance ofmismatched hybrids. Therefore, we conclude that, even though shorterfeet result in lower values for Θ, and even though shorter feet resultin increased C_(T) values, from a strictly thermodynamic viewpoint,there is no reason to believe that shorter foot sequences lead toenhanced discrimination between intended target sequences and unintendedtarget sequences.

L. Furthermore, even though increased bubble circumference also lowersthe value of Θ, it is clear that increasing the circumference of thebubble, though making the formation of hybrids less likely, does notalter the equilibrium ratio of foot hybrids formed from intended targetscompared to foot hybrids formed from unintended hybrids.

M. In terms of classical thermodynamic analysis, it can be shown thatfor any given multi-part primer for which the fraction of molecularcomplex that form foot hybrids is extremely low, the ratio of thefraction of foot hybrids formed with the intended targets (Θ_(m))compared to the fraction of foot hybrids formed with the unintendedtargets (Θ_(w)) is not affected by increasing the circumference of thebubble (which alters ΔS), nor is it affected by decreasing the length ofthe foot (which alters ΔH), but rather, these changes decrease thevalues of both Θ_(w) and Θ_(m), but do not alter the ratio(Θ_(m)/Θ_(w)), which is a function of the difference in the enthalpies(ΔH_(m)−ΔH_(w)). Consequently, from a classical thermodynamic point ofview, the only thing that affects the relative abundance of the intendedhybrids compared to the unintended hybrids is the difference in theirenthalpy values, and this difference is a consequence of the differencein the number of base pairs formed, which is the same no matter what thelength of the foot is. The thermodynamic equation describing the ratio(Θ_(m)/Θ_(w)) is as follows:

(Θ_(m)/Θ_(w))≈e ^(−(ΔHm−ΔHw)/RT)  Equation 6

The experimental results shown in FIG. 10 and FIG. 11 demonstrate thatincreasing the circumference of the bubble and decreasing the length ofthe foot significantly increases the selectivity of the multi-partprimers according to this invention, i.e., these alterations in thedesign of a multi-part primer, though decreasing the abundance of thefoot hybrids, significantly increase the discriminatory ratio,(Θ_(m)/Θ_(w)), as this increase in the discriminatory ratio is evidencedby an increase in the difference in C_(T) values (ΔC_(T)) between theC_(T) obtained with 10⁶ intended target molecules and the C_(T) obtainedwith 10⁶ unintended target molecules. These observations suggest thatthere are additional (perhaps non-thermodynamic reasons) for theextraordinary selectivity of the multi-part primers according to thisinvention.

The explanation for the enhanced selectivity that occurs when themulti-part primers according to this invention are designed so as todecrease the proportion of foot targets that exist at any moment underthe equilibrium conditions of the annealing stages of PCR amplificationassays cannot lie in the discriminatory consequences of ARMS, becausethe degree to which DNA polymerase molecules reject hybrids that do nothave a base pair that includes the 3′-terminal nucleotide of the primeris the same no matter what the abundance of those primers is. Yet, it isclear from the experimental results that an additional discriminatorymechanism is enabling the extraordinary selectivity that occurs when theprimers are designed to rarely form foot hybrids.

While not wishing to be bound by any theory, here is why we believe thatdecreasing the length of the foot and increasing the circumference ofthe bubble enhances selectivity. The explanation lies in our unexpectedrealization that at the relatively high temperatures that exist duringthe annealing stages of a PCR assay, very short foot hybrids only existfor a very short time before they dissociate (measured, perhaps, in tensor hundreds of microseconds). Moreover, the shorter the hybrid, and thelarger the bubble circumference, the shorter is the mean time duringwhich that hybrid exists. We conjecture that the shorter the meanpersistence time of a particular type of hybrid, the more unlikely it isfor a DNA polymerase molecule to encounter one of those hybrids and tothen form a stabilized complex with that hybrid that can undergo chainelongation. The key point here is that whether or not a hybrid will forma stabilized complex with a DNA polymerase molecule is a function of themean persistence time of that hybrid. We believe that the ratio of themean persistence time of a perfectly complementary hybrid formed with aparticular multi-part primer, compared to the mean persistence time of amismatched (shorter) hybrid formed with the same type of multi-partprimer, is greater when the foot length of the primer is decreased andthe bubble circumference of the primer is increased. Thus, morestringent multi-part primer designs (shorter feet, longer bubbles)produce shorter lived hybrids that are considerably less likely to formstabilized hybrids with DNA polymerase molecules. Consequently, shorterfoot hybrids are not only less abundant, they have a lowered chance offorming a stabilized complex with a DNA polymerase molecule, and thisadditional discriminatory property accounts for the extraordinaryselectivity of multi-part primers.

As reported in Example 7, we also investigated the effect of varying thelocation of the interrogating nucleotide in the foot sequence of amulti-part primer according to this invention. We utilized a series ofsix primers: 24-14-6:1:0, 24-14-5:1:1, 24-14-4:1:2, 24-14-3:1:3,24-14-2:1:4, and 24-14-1:1:5. We maintained the length of the anchorsequence, the length of the bridge sequence, and the length of the footsequence (seven nucleotides), only varying the location of theinterrogating nucleotide within the foot sequence. The real-timefluorescence results obtained for each of these primers with 10⁶ copiesof intended target (mutant) and with 10⁶ copies of unintended target(wild-type) are shown in FIG. 12, and the calculated C_(T) values aresummarized in Table 3. The results show that the window ofdiscrimination (ΔC_(T)) between intended target sequences and unintendedtarget sequences increases progressively the closer the location of theinterrogating nucleotide is to the 3′ terminus of the foot. Theseresults indicate that preferred locations for the interrogatingnucleotide are at the 3′ terminus of the foot (enabling ARMSdiscrimination) and at the 3′-penultimate nucleotide of the foot(causing two base pairs to be prevented from forming, rather thanpreventing only one base pair from forming).

As reported in Example 8, we also investigated the shape of the bubbleformed between the bridge sequence of a multi-part primer according tothis invention and the intervening sequence in the intended andunintended target sequences. We altered the “shape of the bubble” bychoosing the relative lengths of these two sequences. In performing theassay, we utilized a series of primers having an anchor sequence 24nucleotides long and having a 5:1:1 foot sequence. We maintained thebubble circumference at 32 nucleotides, but we varied the length of thebridge sequence and the length of the intervening sequence (by alteringthe sequence of the anchor so that upon its hybridization to a templatemolecule, the intervening sequence would be of the desired length). Inaddition to testing a multi-part primer that forms a symmetric bubble,that is, a primer possessing a bridge sequence of 14 nucleotides and ananchor sequence that causes the intervening sequence to be 14nucleotides long (a 14/14 bubble), we tested multi-part primers thatproduced asymmetric bubbles that had relatively longer bridge sequences(an 18/10 bubble and a 16/12 bubble) and that had relatively shorterbridge sequences (a 12/16 bubble and a 10/18 bubble). The real-timefluorescence results obtained for each of these primers with 10⁶ copiesof intended target (mutant) and with 10⁶ copies of unintended target(wild-type) are shown in FIG. 13, and the calculated C_(T) values aresummarized in Table 4. The results show that the window ofdiscrimination (ΔC_(T)) between intended target sequences and unintendedtarget sequences is largest with a symmetric 14/14 bubble, but onlymodestly so. Consequently, our most preferred bubbles are symmetric.

Example 9 reports an experiment utilizing the assay method of Example 4for a different target, B-raf mutation V600E (instead of EGFR mutationL858R) and a 24-14-5:1:1 multi-part primer for that mutation. FIG. 14 isa graph of C_(T) versus the log of the starting number of intendedtarget templates. As can be seen from FIG. 14, this assay provided aΔC_(T) of 23.1 cycles between a sample containing 10⁶ WT templates and asample containing 10⁶ MUT templates in the presence of 10⁶ WT templates,which is even greater than the corresponding ΔC_(T) achieved in Example4.

Example 10 reports another variation, this time utilizing EGFR mutationT790M and PCR amplification using genomic DNA with up to 10,000 copiesof the wild-type target template, and a 24-14-4:1:1 multi-part primer.FIG. 15 is a graph of C_(T) versus the log of the starting number ofintended mutant target templates. As can be seen from FIG. 15, thisassay provided a ΔC_(T) of 12.6 cycles between a sample containing 10⁴WT templates and a sample containing 10⁴ MUT templates in the presenceof 10⁴ WT templates.

Example 11 reports an assay similar to the assay for EGFR mutation L858Rin Example 4 using a different spectrafluorometric thermal cycler, theABI PRISM 7700, the same 24-14-5:1:1 multi-part primer, and plasmid DNA,except that this time the templates were not digested. FIG. 16 is agraph of C_(T) versus the log of the starting number of intended targettemplates. As can be seen from FIG. 16, this assay provided a ΔC_(T) of16.4 cycles between a sample containing 10⁶ WT templates and a samplecontaining 10⁶ MUT templates in the presence of 10⁶ WT templates.

FIG. 17 shows the results of an experiment described in Example 12. Theexperiment was designed to demonstrate the relative contribution ofthermodynamic considerations compared to enzymatic (ARMS-type)considerations in determining the selectivity of the multi-part primersdescribed herein. What we did was to repeat the assay of Example 3 usingnot only the 24-14-5:1:1 primer, but also a truncated 24-14-5:0:0 primerthat omitted the 3′-penultimate and terminal nucleotides. Thus, the footsequence of the latter primer was perfectly complementary to both theintended target sequence and the unintended target sequence. FIG. 17,panel A, compares the amplification of 1,000,000 intended targetsequences to the amplification of 1,000,000 unintended target sequenceswith the 24-14-5:1:1 multi-part primer whose foot/target hybrid isdestabilized at the 3′ end, as is done with ARMS, as well asthermodynamics, to discriminate between the two types of templates. TheC_(T) values for primer 24-14-5:1:1 were 23.1 for the intended targetsequence (curve 1701) and 40.7 for the unintended target sequence (curve1702), giving a ΔC_(T) of 17.6 cycles.

FIG. 17, panel B, compares the amplification of 1,000,000 intendedtarget sequences to the amplification of 1,000,000 unintended targetsequences with the 24-14-5:0:0 primer whose foot/target hybrid is notdestabilized at the 3′ end. The C_(T) values for primer 24-14-5:0:0 were39.7 for the intended target sequence and 39.4 for the unintended targetsequence, giving a ΔC_(T) of −0.3 cycles.

Like truncated primer 24-14-5:0:0, multi-part primer 24-14-5:1:1 forms afoot hybrid with the same five nucleotides in the wild-type template(curve 1702), because this primer's interrogating nucleotide is notcomplementary to the single-nucleotide polymorphism, and the resultingmismatched base pair at the penultimate position of the foot sequenceprevents the adjacent 3′-terminal nucleotide of this primer's footsequence from forming an isolated base pair. There is a difference,however, between the hybrid formed by primer 24-14-5:0:0 with thewild-type template and the hybrid formed by primer 24-14-5:1:1 with thewild-type template, and that difference is that the foot sequence in thehybrid formed by primer 24-14-5:1:1 with the wild-type template has twooverhanging nucleotides caused by the 3′-penultimate mismatch, and istherefore subject to ARMS-type discrimination by DNA polymerase, whereasthe truncated foot sequence in the hybrid formed by primer 24-14-5:0:0with the wild-type template does not have any overhanging 3′-terminalbase pairs, and is therefore not subject to ARMS-type discrimination byDNA polymerase. If ARMS-type discrimination plays a significant role inselectivity when multi-part primers according to this invention areutilized, we would have expected that the C_(T) value of the reactioninvolving primer 24-14-5:0:0 with wild-type templates (curve 1704) wouldhave been lower (i.e., less delayed) than the C_(T) value of thereaction involving primer 24-14-5:1:1 with wild-type templates (curve1702), because ARMS-type discrimination cannot play a role in thereaction involving primer 24-14-5:0:0 with wild-type templates, but canplay a discriminatory role in the reaction involving primer 24-14-5:1:1with wild-type templates. These results suggest that the role ofARMS-type discrimination is absent, or significantly diminished, whenmulti-part primers according to this invention are utilized (perhaps asa result of the extremely short mean persistence time of the foothybrids formed by these highly selective nucleic acid amplificationprimers).

Assays according to this invention may include screening assays lookingfor the presence of any rare target when one of multiple possible raretargets may be present. For such assays a multi-part primer is used foreach possible rare target, but detection need not identify which targetis present. Therefore, SYBR Green dye can be used as the detectionreagent, as can a dual-labeled hybridization probe that signalsindiscriminately, as can a 5′ functional sequence on the primers thatsignals indiscriminately. Assays that employ multi-part primersaccording to this invention include amplification and detection, whichmay include quantitation, of two or more rare target sequencessimultaneously in a single reaction tube, reaction well, or otherreaction vessel, where one needs to identify which target or targets arepresent. The amplification and detection in a single reaction tube oftwo or more rare target sequences that do not have sequence homology andare located in different positions in a genome (for example thesimultaneous detection of rare single-nucleotide polymorphisms locatedin different genes) may include for each different intended targetsequence, a specific, uniquely colored, hybridization probe, such as amolecular beacon probe, a ResonSense® probe, or a 5′-nuclease (TaqMan®)probe that hybridizes to a unique sequence in either strand of theamplified product downstream from the multi-part primer. This appliesnot only to free-floating detector probes, but also to tethered probessuch as molecular beacon probe 409 in FIG. 4. Alternatively, themulti-part primer for each different target sequence may include alabeled hairpin, such as hairpin 404 in FIG. 4. Referring to FIG. 4, twoor more different multi-part primers 103, each specific for a differentrare intended target sequence, and each labeled with a uniquely coloredfluorescent label 408, 413, or 416, can be used to simultaneouslyidentify and quantitate each intended target sequence present in anindividual sample.

5. MULTIPLEX ASSAYS

An especially attractive feature of SuperSelective primers of thisinvention is their potential use in multiplex assays that simultaneouslymeasure the abundance of different rare mutant sequences in the sameclinical sample. The results of these assays can providepatient-specific information to tailor therapy for each individual.

An intriguing multiplex labeling strategy is based on the realizationthat, because there is no relation between the bridge sequence and theintended target sequence, assay designers are free to select adistinctly different bridge sequence for each of the differentSuperSelective primers that are simultaneously present in a multiplexassay. Since the entire sequence of each primer becomes an integral partof the amplicon that is generated when that primer binds to its mutanttarget, the distinctive nucleic acid sequence of the bridge segment canserve as a “serial number” within that amplicon that identifies themutant target from which it was generated.

These identifying bridge sequences can be relatively long (e.g., 20nucleotides in length to assure their uniqueness), and the primers canbe designed to form correspondingly short intervening sequences withinthe template. To simultaneously detect and quantitate different mutanttarget sequences that are present in a clinical sample, a set ofspecific molecular beacon probes (Tyagi et al., (1996) Nat. Biotechnol.14, 303-308, Tyagi et al., (1998) Nat. Biotechnol., 16, 49-53, andBonnet et al., (1999) Proc. Natl. Acad. Sci. USA, 96, 6171-6176) can beincluded in the real-time, gene amplification reactions, each specificfor the complement of the distinctive bridge sequence of one of theSuperSelective primers, and each labeled with a differently coloredfluorophore.

In these reactions, we prefer that the concentration of theSuperSelective forward primers should be limited, and the linear reverseprimers should be present in excess, thereby assuring that the reactionswill not be symmetric, and that the molecular beacons will be able tobind to virtually all of the target amplicons that are synthesized inexcess, without significant competition from less abundant complementaryamplicons (Pierce et al., (2005) Proc. Natl. Acad. Sci. USA, 102,8609-8614). These multiplex assays can even distinguish differentmutations that occur in the same codon, since a SuperSelective primerdesigned to detect a particular mutation will discriminate against aneighboring or alternative mutation in the same way that itdiscriminates against a wild-type target sequence.

Another multiplex strategy is shown in FIG. 18, which is a schematicrepresentation of two multi-part primers according to this inventionthat may be used in a multiplex reaction for two closely relatedintended target sequences.

Where there is sequence homology between or among intended targetsequences in a multiplex assay, a unique sequence can be introduced byutilizing for each different intended target sequence a unique bridgesequence. As explained above in connection with FIG. 2, the reverseprimer copies the entire forward (multi-part) primer into the reverseproduct strand, so in subsequent cycles of amplification the entiremulti-part primer (anchor sequence, bridge sequence, and foot sequence)is complementary to the product made by extension of the reverse primer.In multiplex assays it is important that only one multi-part primer, the“correct” primer that was so copied, hybridizes to and primes thatreverse product strand. It will be appreciated that, therefore, one mustmake the bridge sequence of the “correct” multi-part primer sufficientlydistinct to prevent another multi-part primer from priming that reverseproduct strand (so-called “cross hybridization”). That having been done,a specific, uniquely colored hybridization probe, free-floating ortethered to the primer, that is targeted against the complement of thebridge sequence will signal amplification of only one intended targetand will not signal falsely by hybridizing to the multi-part primeritself. Similarly, only the “correct” multi-part primer with a uniquelycolored hairpin tail (hairpin 405 in FIG. 4) will hybridize to thereverse product strand and signal,

For distinguishing and quantitating the occurrence of different raretarget sequences that are almost identical (differing from each other byonly one or two single-nucleotide polymorphisms) and which occur veryclose to each other within a genome (for example, medically significantvariants of the human K-ras gene, in which different single-nucleotidepolymorphisms can occur within codon 12, each specifying the identity ofa different amino acid in that gene's encoded protein), two or moremulti-part primers can be utilized that possess the structure outlinedin FIG. 18 or in FIG. 19. Turning first to FIG. 18, the top structure103A shows a multi-part primer whose foot sequence 106A is perfectlycomplementary to a specific intended rare target sequence, including thenucleotide in that target sequence that corresponds to complementarynucleotide “g” (the interrogating nucleotide). The lower structure 103Bshows a multi-part primer whose foot sequence 106B is perfectlycomplementary to a different specific rare intended target sequence thatis a variant of the target for foot 106A and which is located at (orvery close to) the position in the genome of the intended targetsequence for foot 106A. In foot sequence 106B, nucleotide “h” is theinterrogating nucleotide that is perfectly complementary to thecorresponding nucleotide in the intended target sequence of foot 106B.In order to be able to simultaneously distinguish, or distinguish andquantitate the abundance of each of these rare target sequences in thesame reaction, primer 103A can be linked to a unique structure 404A,that differs in sequence 405A and 406A and fluorophore label 408A, fromsequence 405B and 406B and fluorophore label 408B in structure 404B ofprimer 103B. When two or more multipart primers, such as primers 103Aand 103B, are used simultaneously for distinguishing and quantitatingsimilar intended rare target sequences at the same (or at a very similarlocation), it is often the case that their respective anchor sequenceswill be identical or very similar (in order to cause the primers to bindto the desired location close to where the variant sequences to bedistinguished occur). However, since there is no relation between abridge sequence of a multi-part primer of this invention and itsintended target sequence, bridge sequence 105A in primer 103A can bechosen so that its nucleotide sequence is different from bridge sequence105B in primer 103B. Here is how two or more multi-part primers of thisinvention can be utilized simultaneously to distinguish and quantitaterare intended target sequences that are alleles of each other and arelocated at the same (or very similar position) in a genome:

Extension of reverse primer 203 (FIG. 2) continues through labeledstructures 404A and 404B, separating quencher 407 from fluorophorelabels 408A and 408B, respectively. As a result, primers 103A and 103Bwill each fluoresce in their unique identifying color when they areincorporated into amplicons, if their fluorescence intensity is measuredin real-time at the end of each chain elongation cycle (in anamplification reaction in which the amplicons become double-stranded,such as in PCR amplifications). Alternatively, primers 103A and 103Bwill each fluoresce in their unique identifying color when theirfluorescence intensity is measured at the end of the annealing stage ofan amplification reaction, because their quencher group 407 becomesseparated from their fluorophore label (408A or 408B) as a consequenceof each primer (103A or 103B) binding to its fully complementarysequence at the 3′ end of those amplicon strands 204 (FIG. 2) whosesynthesis was initiated by the incorporation of the same primer.

FIG. 19 describes primers and probes for a similar assay utilizingfree-floating molecular beacon probes rather than labeled hairpin tails.In FIG. 19 multi-part primer 1903A has foot sequence 1906A that isperfectly complementary to a specific first intended rare targetsequence, including interrogating nucleotide “r”. Multi-part primer1903B has foot sequence 1906B that is perfectly complementary to adifferent specific second rare target sequence that is a variant of thetarget for foot sequence 1906A and which is located at (or very closeto) the position in the genome of the intended target sequence of foot1906A. In foot sequence 1906B, nucleotide “s” is the interrogatingnucleotide. In this embodiment interrogating nucleotide “r” is notcomplementary to either to the second rare target sequence or to thewild-type sequence. And interrogating nucleotide “s” is notcomplementary either to the first rare target sequence or to thewild-type sequence. In order to be able to distinguish amplificationproducts of the two rare target sequences in the same reaction, as wellas to prevent cross hybridization, the sequence of bridge 1905A is madequite different from the sequence of bridge 1905B. Molecular beaconprobe 1907A, comprised of loop 1908A, stem 1909A, fluorophore 1910A andquencher 1911A, has a loop that is specific for the complement of bridgesequence 1905A. Molecular beacon probe 1907B, comprised of loop 1908B,stem 1909B, fluorophore 1910B and quencher 1911B, has a loop that isspecific for the complement of bridge sequence 1905B. Fluorophores 1911Aand 1911B are different colors. Detection by probes 1907A and 1907B canbe either real time or end point.

The key feature that enables simultaneous real-time measurements to bemade of the different amplicons generated from different rare intendedallelic target sequences is that the multi-part primers of thisinvention can be designed to possess quite different sequences in theirlabeled hairpin tails (for example 404A and 404B) and in their bridgesequences (for example 105A and 105B). Consequently, the annealingconditions can be adjusted to assure that each type of primer only bindsto the amplicons whose synthesis was initiated by the same type ofprimer. Moreover, if a particular type of primer were to bind to anon-cognate amplicon, the signaling hairpin at the end of that primerwould not be complementary to the sequence at 3′ end of that amplicon,so no fluorescence would occur. As an alternative to simply utilizingdifferent bridge sequences for each multi-part primer that will besimultaneously present in a reaction, different anchor sequences can beutilized by shortening one or sliding it along the target.Alternatively, different lengths for the bridge sequences (such as 105Aand 105B) would enable the use of different anchor sequences (such as104A and 104B) without significantly altering the selectivity of eachprimer. This will lower the probability of formation of a mismatchedhybrid between primer 103A and non-cognate amplicons containing thepriming sequence for primer 103B, as well as lowering the probability offormation of a mismatched hybrid between primer 103B and non-cognateamplicons containing the priming sequence for primer 103B.

6. ADDITIONAL CONSIDERATIONS FOR DESIGN OF MULTI-PART PRIMERS

Design of multi-part primers according to this invention isstraightforward. We recommend that design be for a particularamplification protocol on a particular instrument, as instruments varyparticularly in their detection and presentation of fluorescence. Asuitable procedure is to choose a design (anchor length, bridge length,and foot length, with the interrogating nucleotide located at either the3′-terminal nucleotide or at the penultimate nucleotide from the 3′ endof the foot. Then, by simply varying the bridge sequence length and thefoot sequence length, in a few trials one can optimize the primer designto achieve the desired large ΔC_(T) between a sample containing intendedtarget and a sample containing unintended target. This involves makingthe primer inefficient for amplifying the intended target sequence.Considerations for design are those discussed above relative to theExamples. In particular, shortening the foot sequence and increasing thesize of the bubble formed by the bridge sequence and the target'sintervening sequence increase the delay in C_(T) with the intendedtarget and increases the ΔC_(T) between a sample containing intendedtarget and a sample containing unintended target.

There are additional considerations in designing multi-part primers ofthis invention. The primer must not prime other sequences that are, ormay be, present in the sample. Conventional computer methods forpreventing that are well known and readily available.

a. Anchor Sequence

The anchor sequence is usually (but not necessarily) perfectlycomplementary to the template sequence, and it usually can be locatedapproximately 14 nucleotides from the 5′ end of the foot sequence andcan usually be 15-40, 15-30 or 20 to 30 (such as 20 to 24) nucleotidesin length. Its length is chosen so that the melting temperature of thehybrid that it forms with the template will be in a suitable range, suchas 66° C. to 72° C. in several of the Examples.

If it turns out that the anchor sequence in a multi-part primer designedto discriminate against a particular polymorphism is not sufficientlyspecific because its target sequence is present elsewhere in the genome,this problem may be solved by designing a multi-part primer thatdiscriminates against the same polymorphism, but binds to thecomplementary target strand.

b. Bridge Sequence

Regarding the bridge sequence, we recommend checking for and, ifnecessary, eliminating transient hybridization events that may occur ifthat sequence can form low-Tm hybrids with the target, thereby reducingits effective length. Also, the effect of the bridge can be modified byadjusting the rigidity of the bridge sequence, as different nucleotidesequences have somewhat different rigidities. See Goddard et al. (2000)Phys. Rev. Lett. 85:2400-2403.

In one example, the bridge sequence can be approximately at least 6(e.g., 7, 8, 9, 10, 11, 12, 13, 14, 15, or 20) nucleotides in length.Its nucleotide sequence can be chosen to ensure that, under annealingconditions: (i) it does not hybridize to the corresponding “interveningsequence” in the template strand (which is located between the foottarget sequence and the anchor target sequence); (ii) it does nothybridize to any sequence in the human genome; (iii) it does not formany secondary structures under assay conditions that would effectivelyshorten its length; and (iv) it does not hybridize to the conventionalreverse primer used to prime the synthesis of the complementary templatestrand. In addition, if the intervening sequence in the template strandmight form secondary structures under assay conditions that effectivelyshorten its length, the length of the bridge sequence can be increasedand the length of the intervening sequence can be decreased by acorresponding number of nucleotides (accomplished by selecting an anchortarget sequence that is closer to the foot target sequence by the samenumber of nucleotides).

The realization that the bridge sequence can be chosen to be relativelyshort or relatively long, and the realization that the probe designercan chose any arbitrary sequence for the bridge segment, opens up aplethora of functional possibilities for the design of theSuperSelective primers of this invention.

For example, if the sequence of a putative intervening sequence thatoccurs naturally in the template is such that it might form a secondarystructure under assay conditions, the primer can be designed so as tocreate a relatively small intervening sequence in the primer-templatehybrid, thereby disrupting the formation of the secondary structure, andthe primer's bridge sequence can be chosen to be of a relatively longerlength, thereby preserving the selectivity of the assay (see the resultsshown in Table 4). Moreover, primer function can be fine-tuned, byselecting a sequence for the bridge that takes into account differencesin the flexibility of the intervening sequence and the bridge sequence.

Furthermore, the choice of an appropriate bridge sequence for aSuperSelective primer apparently suppresses the occurrence of falseamplicons, such as primer-dimers. Unlike the design of conventionallinear primers (whose sequence is determined by the template to which itbinds), an arbitrary sequence is used for the bridge segment. We takecare to select a bridge sequence that: (i) does not form secondarystructures; (ii) is unrelated to the sequence of the template, thesequence of the genomic DNA, and the sequence of the conventionalreverse primer; and that, (iii) when incorporated into the full-lengthprimer, does not enable primer self-hybridization.

c. Role of the Bubble Formed by the Bridge Sequence and the InterveningSequence

Within the acceptable ranges described above, the greater thecircumference of the bubble formed by the hybridization of aSuperSelective primer to an original template molecule, the greater isthe suppression of wild-type amplicon synthesis relative to thesuppression of mutant amplicon synthesis (see for example, FIG. 11).From a thermodynamic point of view, larger bubbles should reduce theequilibrium abundance of both the wild-type hybrids and the mutanthybrids, but should not alter their relative abundance. However, from akinetic point of view, it is appropriate to consider the forces thatimpinge upon the bubble that connects the foot hybrid to the targethybrid, because the bubble is subject to random Brownian motions of thewater molecules in the reaction mixture. This creates a force that hasthe potential to pull the foot hybrids apart. The greater thecircumference of the bubble, the greater is this potentially disruptiveforce. Moreover, mismatched wild-type hybrids, which are weaker thanperfectly complementary mutant hybrids, are more likely to be pulledapart.

Thus, mismatched wild-type hybrids, not only exist for a shorter lengthof time due to their lower stability, they are also more easily pulledapart by the random forces that impinge on the bubble. We thereforebelieve that the extraordinary selectivity of SuperSelective primersarises from both thermodynamic factors that affect hybrid stability, andfrom kinetic factors that affect the mean persistence time of theresulting hybrids.

d. Foot Sequence

The foot sequence is located at the 3′ end of the primer; it iscomplementary to the region of the template strand where there is atleast one nucleotide difference between the intended target sequence andits closely related unintended target sequence such as asingle-nucleotide polymorphism is located; and it is usually sevennucleotides in length. The “interrogating nucleotide” in the footsequence may be located at the penultimate position from the 3′ end ofthe foot sequence, or at the 3′ end of the foot sequence. The length ofthe foot sequence can be modified to improve selectivity. The footsequence can be shorter (six or even five nucleotides in length),especially if it has a high G-C content. If the interrogating nucleotidewould form a G:T base pair with the wild-type template strand, it isdesirable to design the primer so that it binds to the complementarytemplate strand, instead.

If the foot sequence is hybridized to the target sequence, and if theDNA polymerase is able to form a functional complex with that hybridbefore the hybrid falls apart, then the extension of the foot sequencecan be catalyzed by the DNA polymerase to generate an amplicon. It willbe appreciated that short foot sequences, for example, 6 or 7nucleotides in length, generally are so short that they arecomplementary to sequences that occur at a large number of differentlocations within the nucleic acids that may be present in a sample beingtested, for example in genomic DNA from human cells. However, the footsequence is so short, and consequently has a melting temperature, Tm,that is so extremely low under the conditions used for amplification,such as the conditions that are used in PCR assays, that the footsequence will not form a hybrid with any perfectly complementarysequence in the nucleic acid sample being tested, unless the anchorsequence of the primer has first hybridized to a location within thenucleic acid being tested that is only a few nucleotides away from thedesired target sequence.

Once designed in the manner disclosed herein, primer sequences can beexamined with the aid of any suitable computer program, such as theOligoAnalyzer computer program (Integrated DNA Technologies, Coralville,Iowa), to ensure that under assay conditions they are unlikely to forminternal hairpin structures or self-dimers, and to ensure that they donot form heterodimers with the conventional reverse primers.

7. KITS

This invention further includes reagent kits containing reagents forperforming the above-described amplification methods, includingamplification and detection methods. To that end, one or more of thereaction components for the methods disclosed herein can be supplied inthe form of a kit for use in the detection of a target nucleic acid. Insuch a kit, an appropriate amount of one or more reaction components isprovided in one or more containers or held on a substrate (e.g., byelectrostatic interactions or covalent bonding).

The kit described herein includes one or more of the primers describedabove. The kit can include one or more containers containing one or moreprimers of the invention. A kit can contain a single primer in a singlecontainer, multiple containers containing the same primer, a singlecontainer containing two or more different primers of the invention, ormultiple containers containing different primers or containing mixturesof two or more primers. Any combination and permutation of primers andcontainers is encompassed by the kits of the invention

The kit also contains additional materials for practicing theabove-described methods. In some embodiments, the kit contains some orall of the reagents, materials for performing a method that uses aprimer according to the invention. The kit thus may comprise some or allof the reagents for performing a PCR reaction using the primer of theinvention. Some or all of the components of the kits can be provided incontainers separate from the container(s) containing the primer of theinvention. Examples of additional components of the kits include, butare not limited to, one or more different polymerases, one or moreprimers that are specific for a control nucleic acid or for a targetnucleic acid, one or more probes that are specific for a control nucleicacid or for a target nucleic acid, buffers for polymerization reactions(in 1× or concentrated forms), and one or more dyes or fluorescentmolecules for detecting polymerization products. The kit may alsoinclude one or more of the following components: supports, terminating,modifying or digestion reagents, osmolytes, and an apparatus fordetecting a detection probe.

The reaction components used in an amplification and/or detectionprocess may be provided in a variety of forms. For example, thecomponents (e.g., enzymes, nucleotide triphosphates, probes and/orprimers) can be suspended in an aqueous solution or as a freeze-dried orlyophilized powder, pellet, or bead. In the latter case, the components,when reconstituted, form a complete mixture of components for use in anassay.

A kit or system may contain, in an amount sufficient for at least oneassay, any combination of the components described herein, and mayfurther include instructions recorded in a tangible form for use of thecomponents. In some applications, one or more reaction components may beprovided in pre-measured single use amounts in individual, typicallydisposable, tubes or equivalent containers. With such an arrangement,the sample to be tested for the presence of a target nucleic acid can beadded to the individual tubes and amplification carried out directly.The amount of a component supplied in the kit can be any appropriateamount, and may depend on the target market to which the product isdirected. General guidelines for determining appropriate amounts may befound in, for example, Joseph Sambrook and David W. Russell, MolecularCloning: A Laboratory Manual, 3rd edition, Cold Spring Harbor LaboratoryPress, 2001; and Frederick M. Ausubel, Current Protocols in MolecularBiology, John Wiley & Sons, 2003.

The kits of the invention can comprise any number of additional reagentsor substances that are useful for practicing a method of the invention.Such substances include, but are not limited to: reagents (includingbuffers) for lysis of cells, divalent cation chelating agents or otheragents that inhibit unwanted nucleases, control DNA for use in ensuringthat primers, the polymerase and other components of reactions arefunctioning properly, DNA fragmenting reagents (including buffers),amplification reaction reagents (including buffers), and wash solutions.The kits of the invention can be provided at any temperature. Forexample, for storage of kits containing protein components or complexesthereof in a liquid, it is preferred that they are provided andmaintained below 0° C., preferably at or below −20° C., or otherwise ina frozen state.

The container(s) in which the components are supplied can be anyconventional container that is capable of holding the supplied form, forinstance, microfuge tubes, ampoules, bottles, or integral testingdevices, such as fluidic devices, cartridges, lateral flow, or othersimilar devices. The kits can include either labeled or unlabelednucleic acid probes for use in amplification or detection of targetnucleic acids. In some embodiments, the kits can further includeinstructions to use the components in any of the methods describedherein, e.g., a method using a crude matrix without nucleic acidextraction and/or purification.

The kits can also include packaging materials for holding the containeror combination of containers. Typical packaging materials for such kitsand systems include solid matrices (e.g., glass, plastic, paper, foil,micro-particles and the like) that hold the reaction components ordetection probes in any of a variety of configurations (e.g., in a vial,microtiter plate well, microarray, and the like).

8. ADDITIONAL DEFINITIONS

As used herein, the term “target nucleic acid” or “target sequence”refers to a nucleic acid containing a target nucleic acid sequence. Atarget nucleic acid may be single-stranded or double-stranded, and oftenis DNA, RNA, a derivative of DNA or RNA, or a combination thereof. A“target nucleic acid sequence,” “target sequence” or “target region”means a specific sequence comprising all or part of the sequence of asingle-stranded nucleic acid. A target sequence may be within a nucleicacid template, which may be any form of single-stranded ordouble-stranded nucleic acid. A template may be a purified or isolatednucleic acid, or may be non-purified or non-isolated.

As used herein the term “amplification” and its variants includes anyprocess for producing multiple copies or complements of at least someportion of a polynucleotide, said polynucleotide typically beingreferred to as a “template.” The template polynucleotide can be singlestranded or double stranded. Amplification of a given template canresult in the generation of a population of polynucleotide amplificationproducts, collectively referred to as an “amplicon.” The polynucleotidesof the amplicon can be single stranded or double stranded, or a mixtureof both. Typically, the template will include a target sequence, and theresulting amplicon will include polynucleotides having a sequence thatis either substantially identical or substantially complementary to thetarget sequence. In some embodiments, the polynucleotides of aparticular amplicon are substantially identical, or substantiallycomplementary, to each other; alternatively, in some embodiments thepolynucleotides within a given amplicon can have nucleotide sequencesthat vary from each other. Amplification can proceed in linear orexponential fashion, and can involve repeated and consecutivereplications of a given template to form two or more amplificationproducts. Some typical amplification reactions involve successive andrepeated cycles of template-based nucleic acid synthesis, resulting inthe formation of a plurality of daughter polynucleotides containing atleast some portion of the nucleotide sequence of the template andsharing at least some degree of nucleotide sequence identity (orcomplementarity) with the template. In some embodiments, each instanceof nucleic acid synthesis, which can be referred to as a “cycle” ofamplification, includes creating free 3′ end (e.g., by nicking onestrand of a dsDNA) thereby generating a primer and primer extensionsteps; optionally, an additional denaturation step can also be includedwherein the template is partially or completely denatured. In someembodiments, one round of amplification includes a given number ofrepetitions of a single cycle of amplification. For example, a round ofamplification can include 5, 10, 15, 20, 25, 30, 35, 40, 50, or morerepetitions of a particular cycle. In one exemplary embodiment,amplification includes any reaction wherein a particular polynucleotidetemplate is subjected to two consecutive cycles of nucleic acidsynthesis. The synthesis can include template-dependent nucleic acidsynthesis.

The term “primer” or “primer oligonucleotide” refers to a strand ofnucleic acid or an oligonucleotide capable of hybridizing to a templatenucleic acid and acting as the initiation point for incorporatingextension nucleotides according to the composition of the templatenucleic acid for nucleic acid synthesis. “Extension nucleotides” referto any nucleotide capable of being incorporated into an extensionproduct during amplification, i.e., DNA, RNA, or a derivative if DNA orRNA, which may include a label.

“Hybridization” or “hybridize” or “anneal” refers to the ability ofcompletely or partially complementary nucleic acid strands to cometogether under specified hybridization conditions (e.g., stringenthybridization conditions) in a parallel or preferably antiparallelorientation to form a stable double-stranded structure or region(sometimes called a “hybrid”) in which the two constituent strands arejoined by hydrogen bonds. Although hydrogen bonds typically form betweenadenine and thymine or uracil (A and T or U) or cytosine and guanine (Cand G), other base pairs may form (e.g., Adams et al., The Biochemistryof the Nucleic Acids, 11th ed., 1992).

The term “stringent hybridization conditions” or “stringent conditions”means conditions in which a probe or oligomer hybridizes specifically toits intended target nucleic acid sequence and not to another sequence.Stringent conditions may vary depending well-known factors, e.g., GCcontent and sequence length, and may be predicted or determinedempirically using standard methods well known to one of ordinary skillin molecular biology (e.g., Sambrook, J. et al., 1989, MolecularCloning, A Laboratory Manual, 2nd ed., Ch. 11, pp. 11.47-11.57, (ColdSpring Harbor Laboratory Press, Cold Spring Harbor, N.Y.)).

As disclosed herein, a number of ranges of values are provided. It isunderstood that each intervening value, to the tenth of the unit of thelower limit, unless the context clearly dictates otherwise, between theupper and lower limits of that range is also specifically disclosed.Each smaller range between any stated value or intervening value in astated range and any other stated or intervening value in that statedrange is encompassed within the invention. The upper and lower limits ofthese smaller ranges may independently be included or excluded in therange, and each range where either, neither, or both limits are includedin the smaller ranges is also encompassed within the invention, subjectto any specifically excluded limit in the stated range. Where the statedrange includes one or both of the limits, ranges excluding either orboth of those included limits are also included in the invention.

The term “about” generally refers to plus or minus 10% of the indicatednumber. For example, “about 10%” may indicate a range of 9% to 11%, and“about 1” may mean from 0.9-1.1. Other meanings of “about” may beapparent from the context, such as rounding off, so, for example “about1” may also mean from 0.5 to 1.4.

EXAMPLES Example 1 EGFR Mutation L858R and a Conventional Linear Primer

Two PCR amplification and detection assays were carried out using as atemplate either a plasmid DNA containing EGFR mutation L858R or aplasmid DNA containing the corresponding wild-type sequence, whichdiffered from each other by a single-nucleotide polymorphism.Conventional forward and reverse primers were used to generate adouble-stranded amplification product 49 nucleotides long. The forwardprimer (FP) was a conventional primer, containing the interrogatingnucleotide near the middle of the primer sequence. The reverse primer(RP) was a conventional primer that was perfectly complementary to bothtarget sequences. The primer sequences and the intended target sequencepossessing the mutant allele (MUT), were as follows:

FP:  (SEQ ID No. 1) 5′-ATTTTGGGC G GGCCAAACTGC-3′ MUT: (SEQ ID No. 2)3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAACCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′ RP:  (SEQ ID No. 3)5′-GCATGGTATTCTTTCTCTTCCGCA-3′

In the forward primer sequence, the nucleotide that is complementary tothe mutant target template, but mismatched to the wild-type template, isbold, underlined, and larger. In the mutant target sequence, the bindingsite for the forward primer is underlined, and the sequence of thereverse primer is underlined. In addition, in the mutant targetsequence, the nucleotide specific to the mutant is bolded, underlined,and larger. Using Integrated DNA Technologies' SciTools program forcalculating the melting temperatures of DNA hybrids (specifyingparameters: [oligo]=0.06 μM; [Na⁺]=60 mM; [Mg²⁺]=3 mM; [dNTPs]=0.25 mM),the calculated Tm of the forward primer bound to the mutant allele is67.5° C., and the calculated Tm for the reverse primer is 64.0° C.

Plasmids were prepared by inserting a 115 base pair EGFR gene fragment,containing either the EGFR L858R mutation or the corresponding EGFRwild-type sequence, into a pGEM-11Zf(+) vector (Promega). Mutant andwild-type plasmid DNAs were digested with the restriction endonucleaseMse I (New England Biolabs). The digestion mixture contained 10 unitsMse I and 4 μg of mutant or wild-type genomic DNA in a 20-μl volume thatcontained 5 mM KAc, 2 mM Tris-Ac (pH 7.9), 1 mM MgAc, 1% bovine serumalbumin, and 100 μM dithiothreitol. The reactions were incubated for 120min at 37° C., followed by an incubation for 20 min at 65° C. toinactivate the enzyme.

PCR amplifications were performed in a 30-μl volume containing 50 mMKCl, 10 mM Tris-HCl (pH 8.0), 3 mM MgCl₂, 1.5 Units AmpliTaq Gold DNApolymerase (Life Technologies), 250 μM each of the fourdeoxyribonucleoside triphosphates (dNTPs), 60 nM of each primer, and1×SYBR® Green (Life Technologies). In this series, reaction mixturescontained either 10⁶ copies of the mutant template (MUT) or 10⁶ copiesof wild-type template (WT). Amplifications were carried out using 0.2 mlpolypropylene PCR tubes (white) in a Bio-Rad IQ5 spectrofluorometricthermal cycler. The thermal-cycling profile was 10 min at 95° C.,followed by 60 cycles of 94° C. for 15 sec, 60° C. for 15 sec, and 72°C. for 20 sec. SYBR® Green fluorescence intensity was measured at theend of each chain elongation stage (72° C.).

Real-time fluorescence results, that is, SYBR Green® fluorescenceintensity as a function

of the number of amplification cycles completed, are shown in FIG. 5,where curve 501 is the reaction containing 10⁶ MUT templates and curve502 is the reaction containing 10⁶ WT templates. The assay instrumentautomatically calculates the threshold cycle (C_(T)) for each reaction.These values were 20.0 (curve 501) and 19.7 (curve 502). In the upperleft-hand corner of the graph is a schematic representation of theconventional forward primer (straight line) with the interrogatingnucleotide (circle) in the middle.

Example 2 EGFR Mutation L858R and a Conventional Linear Primer with a3′-Terminal Interrogating Nucleotide

A PCR amplification and detection assay was carried out using the mutant(MUT) and wild-type (WT) templates described in Example 1. In thisexperiment, the forward primer is an “ARMS Primer,” that is, a primerperfectly complementary to the mutant template, but possessing a3′-terminal mismatch to the WT template, that is, possessing aninterrogating nucleotide at the 3′ end of the priming sequence. We usedthe same reverse primer as in Example 1. The primer sequences and theintended target sequence possessing the mutant allele (MUT), were asfollows:

FP:  (SEQ ID No. 4) 5′-CAAGATCACAGATTTTGGGC G -3′ MUT: (SEQ ID No. 2)3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAA CCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′ RP:  (SEQ ID No. 3)5′-GCATGGTATTCTTTCTCTTCCGCA-3′

In the forward primer sequence, the nucleotide that is complementary tothe mutant target template, but mismatched to the wild-type template, isbolded, underlined, and larger. In the mutant target sequence, thebinding site for the forward primer is underlined, and the sequence ofthe reverse primer is underlined. In addition, in the mutant targetsequence, the nucleotide specific to the mutant is bolded, underlined,and larger. Using Integrated DNA Technologies' SciTools program forcalculating the melting temperatures of DNA hybrids (specifyingparameters: [oligo]=0.06 μM; [Na⁺]=60 mM; [Mg²⁺]=3 mM; [dNTPs]=0.25 mM),the calculated Tm of the forward primer bound to the mutant allele is60.7° C., and the calculated Tm for the reverse primer is 64.0° C.

PCR amplification was carried out as described in Example 1. Real-timefluorescence results, that is, SYBR Green® fluorescence intensity as afunction of the number of amplification cycles completed, are shown inFIG. 6, Panel A, where curve 601 is the reaction starting with 10⁶ MUTtemplates and curve 602 is the reaction starting with 10⁶ WT templates.The assay instrument automatically calculates the threshold cycle(C_(T)) for each curve. Those values were 19.4 (curve 601) and 30.4(curve 602), resulting in a ΔC_(T) of 11 cycles. In the upper left-handcorner of the graph is a schematic representation of the conventionalforward primer (straight line) with the interrogating nucleotide(circle) located at the 3′ end of the primer.

The experiment described above was repeated with a forward primer thatpossessed the interrogating nucleotide at the penultimate position fromits 3′ end (we added a G to the 3′ end of the primer and removed the5′-terminal C to maintain primer length). The sequence of the resultingforward primer was:

FP: 5′-AAGATCACAGATTTTGGGCGG-3′ (SEQ ID No. 5)

Using Integrated DNA Technologies' SciTools program, and the samereaction conditions described above, the calculated Tm of the forwardprimer bound to the mutant allele was 61.9° C.

Real-time fluorescence results, that is, SYBR Green® fluorescenceintensity as a function of the number of amplification cycles completed,are shown in FIG. 6, Panel B, where curve 603 is the reaction startingwith 10⁶ MUT templates and curve 604 is the reaction starting with 10⁶WT templates. The machine-calculated C_(T) values were 19.1 (curve 603)and 27.8 (curve 604), resulting in a ΔC_(T) of 8.8 cycles. In the upperleft-hand corner of the graph is a schematic representation of theconventional forward primer (straight line) with the interrogatingnucleotide (circle) located at the penultimate position from the 3′ endof the primer.

Example 3 EGFR Mutation L858R and a 24-14-5:1:1 Multi-part Primer(Real-time Data)

Two PCR amplification and detection assays were carried out using themutant (MUT) and wild-type (WT) template described in Example 1. In thisexperiment, the forward primer (FP) is a multi-part primer according tothis invention. We used the same reverse primer as in Example 1.

In our nomenclature, the multi-part primer used in this example isreferred to as a 24-14-5:1:1 primer, referring to an anchor sequencethat is 24 nucleotides long, a bridge sequence that is 14 nucleotideslong, and a foot sequence that is seven nucleotides long (comprising,from the 5′ end of the foot, five nucleotides complementary to both theMUT and WT targets, one interrogating nucleotide that is notcomplementary to the corresponding nucleotide in the WT target, but thatis complementary to the corresponding nucleotide in the MUT target, and,finally, one nucleotide complementary to both targets. Because theinterrogating nucleotide is located one nucleotide inboard of the 3′ endof the primer, we refer to this nucleotide as being located at the“3′-penultimate position.” Comparing the bridge sequence to the regionof the target sequence lying between the binding sequence of the anchorand the binding sequence of the foot, which we call the “interveningsequence,” one sees that the intervening sequence in this example isfourteen nucleotides long, the same length as the bridge sequence. Thesequence of the bridge sequence is chosen so that it is notcomplementary to the intervening sequence, in order to prevent thehybridization of the bridge sequence to the intervening sequence duringprimer annealing. Instead of annealing to each other, the bridgesequence and the intervening sequence form a single-stranded “bubble”when both the anchor sequence and the foot sequence are hybridized tothe template. The “circumference of the bubble” is defined as the sum ofthe number of nucleotides in the bridge sequence plus the number ofnucleotides in the intervening sequence plus the anchor sequence's 3′nucleotide and its complement plus the foot sequence's 5′-terminalnucleotide and its complement. Consequently, the circumference of thebubble formed by the binding of the multi-part primer in this example tothe template molecules used in this example is 14+14+2+2, which equals32 nucleotides in length.

The primer sequences and the intended target sequence possessing themutant allele (MUT), were as follows:

Primer 24-14-5:1:1 Anchor Bridge Foot FP:  (SEQ ID No. 6)5′-CTGGTGAAAACACCGCAGCATGTCGCACGAGTGAGCCCTGGGC G G-3′ MUT: (SEQ ID No. 2) 3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAACCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′ RP:  (SEQ ID No. 3)5′-GCATGGTATTCTTTCTCTTCCGCA-3′

In the multi-part forward primer, the bridge sequence is underlined, andthe interrogating nucleotide in the foot sequence is bolded, underlined,and larger. In the mutant target sequence, the binding sequence for theforward primer's anchor and for the forward primer's foot areunderlined, and the sequence of the reverse primer is underlined. Inaddition, in the mutant target sequence, the nucleotide specific to themutant is bolded, underlined, and larger. Using Integrated DNATechnologies' SciTools program for calculating the melting temperaturesof DNA hybrids (specifying parameters: [oligo]=0.06 μM; [Na⁺]=60 mM;[Mg²⁺]=3 mM; [dNTPs]=0.25 mM), the Tm for the binding of the anchorsequence to a template is 66.9° C., and the Tm for the binding of theentire multi-part primer to the resulting complementary amplicon is79.9° C.

PCR amplifications were carried out as described in Example 1. Real-timefluorescence results, that is, SYBR Green® fluorescence intensity as afunction of the number of amplification cycles completed, are shown inFIG. 7, where curve 701 is the reaction starting with 10⁶ MUT templatesand curve 702 is the reaction starting with 10⁶ WT templates. The assayinstrument automatically calculates the threshold cycle (C_(T)) for eachreaction. These values were 22.9 (curve 701) and 41.1 (curve 702),resulting in a ΔC_(T) of 18.2 cycles. In the upper left-hand corner ofthe graph is a schematic representation of the multi-part primer (thebridge sequence being the semicircle) with the interrogating nucleotide(circle) located at the penultimate position from 3′ end of the primer.

Example 4 EGFR Mutation L858R and a 24-14-5:1:1 Multi-Part Primer(Selective Amplification)

A series of PCR amplification and detection assays was carried out usingthe same multi-part primer, reverse primer, intended target (MUT), andunintended target (WT) described in Example 3. The amplifications werecarried out as described in Example 3. Real-time fluorescence results,that is, SYBR Green® fluorescence intensity as a function of the numberof amplification cycles completed, are shown in FIG. 8, where curve 801is the reaction starting with 10⁶ WT templates, and curves 802-807 arethe dilution series where each reaction contained 10⁶ WT templates pluseither 10⁶, 10⁵, 10⁴, 10³, 10², or 10¹ MUT templates, respectively. Theassay instrument automatically calculates the threshold cycle (C_(T))for each reaction. Those values were 41.1 (curve 801), 23.3 (curve 802),26.8 (curve 803), 30.5 (curve 804), 33.8 (curve 805), 37.0 (curve 806),and 39.2 (curve 807). In the upper left-hand corner of the graph is aschematic representation of the multi-part primer (the bridge sequencebeing the semicircle) with the interrogating nucleotide (circle) locatedat the penultimate position from 3′ end of the primer.

FIG. 9 is a graph of the C_(T) values observed for each reaction thatcontained MUT templates (obtained from curves 802 through 807 in FIG. 8)as a function of the logarithm of the number of MUT templates present inthat reaction. Line 901 is a linear correlation fit to the data points.Dashed line 902 identifies the C_(T) value for the amplificationinitiated with 10⁶ WT templates and no MUT templates.

Example 5 EGFR Mutation L858R and the Effect of Decreasing theMulti-Part Primer Foot Length

The experiment described in Example 4 was repeated using the same24-14-5:1:1 primer (SEQ. ID No. 6) possessing a foot sequence that isseven-nucleotides long; and also using two additional multi-part primersof the same design, except that the foot sequence of one of theadditional primers was one nucleotide longer (24-14-6:1:1), and the footsequence of the other additional primer was one nucleotide shorter(24-14-4:1:1). In all three cases, the anchor sequence was 24nucleotides long, the bridge sequence was 14 nucleotides long, and thetarget's intervening sequence was 14 nucleotides long, creating a bubblecircumference of 32 nucleotides in all cases. Furthermore, in all threecases, the interrogating nucleotide was located at the 3′-penultimateposition in the foot of the primer. Primer sequences and their intendedtarget sequence (MUT), were as follows:

Primer 24-14-4:1:1 Anchor Bridge Foot FP:  (SEQ ID No. 7)5′-TGGTGAAAACACCGCAGCATGTCACACGAGTGAGCCCCGGGC G G-3′ MUT: (SEQ ID No. 2) 3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAACCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′Primer 24-14-5:1:1 Anchor Bridge Foot FP:  (SEQ ID No. 6)5′-CTGGTGAAAACACCGCAGCATGTCGCACGAGTGAGCCCTGGGC G G-3′ MUT: (SEQ ID No. 2) 3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAACCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′Primer 24-14-6:1:1 Anchor Bridge Foot FP:  (SEQ ID No. 8)5′-ACTGGTGAAAACACCGCAGCATGTTGGAGCTGTGAGCCTTGGGC G G-3′ MUT: (SEQ ID No. 2) 3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAACCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′ Reverse Primer RP: (SEQ ID No. 3) 5′-GCATGGTATTCTTTCTCTTCCGCA-3′

In the multi-part forward primers, the bridge sequence is underlined,and the interrogating nucleotide in the foot sequence is bolded,underlined, and larger. In the mutant target sequence, the bindingsequence for the forward primer's anchor and for the forward primer'sfoot are underlined, and the sequence of the reverse primer isunderlined. In addition, in the mutant target sequence, the nucleotidespecific to the mutant is bolded, underlined, and larger. UsingIntegrated DNA Technologies' SciTools program for calculating themelting temperatures of DNA hybrids (specifying parameters: [oligo]=0.06μM; [Na⁺]=60 mM; [Mg²]=3 mM; [dNTPs]=0.25 mM); the Tm for the binding ofthe 24-14-4:1:1 anchor sequence to a template is 68.1° C., and the Tmfor the binding of the entire multi-part primer to the resultingcomplementary amplicon is 80.3° C.; the Tm for the binding of the24-14-5:1:1 anchor sequence to a template is 66.9° C., and the Tm forthe binding of the entire multi-part primer to the resultingcomplementary amplicon is 79.9° C.; and the Tm for the binding of the24-14-6:1:1 anchor sequence to a template is 68.1° C., and the Tm forthe binding of the entire multi-part primer to the resultingcomplementary amplicon is 79.4° C.

For each of the three multi-part primer designs, a series of PCRamplification and detection assays was carried out as described inExample 4, utilizing a dilution series starting with 10⁶ WT templatesplus 10⁶, 10⁵, 10⁴, 10³, 10², or 10¹ copies of the MUT template,respectively. The assay instrument automatically calculates thethreshold cycle (C_(T)) for each reaction. The C_(T) values calculatedfrom the real-time data for each reaction (not shown) are listed inTable 1, along with the calculated C_(T) value for reactions initiatedwith 10⁶ WT templates and no MUT templates.

TABLE 1 Threshold Cycles (C_(T)) Observed for Reactions ContainingDifferent Numbers of Intended Targets Primer 10⁶ 10⁵ 10⁴ 10³ 10² 10¹ 024-14-4:1:1 27.5 30.7 34.2 37.1 40.3 44.6 42.0 24-14-5:1:1 23.3 26.630.4 33.4 37.0 38.8 41.1 24-14-6:1:1 21.2 24.6 27.9 32.0 34.9 35.6 37.5

FIG. 10 is a set of graphs showing the C_(T) values observed (for eachset of reactions containing the same primer) as a function of thelogarithm of the number of MUT templates present in each reaction. Line1001 is a linear correlation fit to the C_(T) values for the primerpossessing a six-nucleotide-long foot sequence (4:1:1); line 1002 is alinear correlation fit to the C_(T) values for the primer possessing aseven-nucleotide-long foot sequence (5:1:1); and line 1003 is a linearcorrelation curve fit to the C_(T) values for the primer possessing aseven-nucleotide-long foot sequence (6:1:1). When the 24-14-6:1:1 primerwas used, the lower abundance MUT template samples gave C_(T) valuesthat occurred somewhat earlier than predicted, suggesting the presenceof a few obscuring amplicons generated from the abundant WT templates inthe sample.

These results demonstrate that the use of a multi-part primer possessinga shorter foot sequence, such as primer 24-14-5:1:1, reduces thisproblem, and the use of a primer possessing the shortest foot sequence,such as primer 24-14-4:1:1, virtually eliminates this problem, enablingthe detection and quantitation of as few as 10 intended templatemolecules in the presence of 1,000,000 unintended template molecules.

Example 6 EGFR Mutation L858R and the Effect of Increasing theMulti-Part Primer Bubble Circumference

The experiment described in Example 4 was repeated using the same24-14-5:1:1 primer (SEQ. ID No. 6) possessing a bridge sequence14-nucleotides long that creates an intervening sequence when hybridizedto its template that is also 14-nucleotides long; and also using twoadditional multi-part primers of the same design, except that the bridgesequence of one of the additional primers was 18-nucleotides long(24-18-5:1:1), and the bridge sequence of the other additional primerwas 10-nucleotides long (24-10-5:1:1). In all three cases, the anchorsequence was 24-nucleotides long, the foot sequence was 5:1:1, and thechoice of the anchor sequence was such that the intervening sequencecreated when the primer binds to its template was the same length as theprimer's bridge sequence. Consequently, the bubble circumferences formedby this series of three multi-part primers are 24, 32, and 40nucleotides in length, respectively. Furthermore, in all three cases,the interrogating nucleotide was located at the 3′-penultimate positionin the foot of the primer. Primer sequences and the intended targetsequence (MUT), were as follows:

Primer 24-10-5:1:1 Anchor Bridge Foot FP: (SEQ ID No. 10)5′-TGAAAACACCGCAGCATGTCAAGACACTCAGCCCTGGGC G G-3′ MUT:  (SEQ ID No. 2)3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAACCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′Primer 24-14-5:1:1 Anchor Bridge Foot FP:  (SEQ ID No. 6)5′-CTGGTGAAAACACCGCAGCATGTCGCACGAGTGAGCCCTGGGC G G-3′ MUT: (SEQ ID No. 2) 3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAACCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′Primer 24-18-5:1:1 Anchor Bridge Foot FP:  (SEQ ID No. 9)5′-CGTACTGGTGAAAACACCGCAGCACTGACGACAAGTGAGCCCTGGG C G G-3′ MUT:(SEQ ID No. 2) 3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAACCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′ Reverse Primer RP:(SEQ ID No. 3) 5′-GCATGGTATTCTTTCTCTTCCGCA-3′

In the multi-part forward primers, the bridge sequence is underlined,and the interrogating nucleotide in the foot sequence is bolded,underlined, and larger. In the mutant target sequence, the bindingsequence for the forward primer's anchor and for the forward primer'sfoot are underlined, and the sequence of the reverse primer isunderlined. In addition, in the mutant target sequence, the nucleotidespecific to the mutant is bolded, underlined, and larger. UsingIntegrated DNA Technologies' SciTools program for calculating themelting temperatures of DNA hybrids (specifying parameters: [oligo]=0.06μM; [Na⁺]=60 mM; [Mg²⁺]=3 mM; [dNTPs]=0.25 mM); the Tm for the bindingof the 24-10-5:1:1 anchor sequence to a template is 66.3° C., and the Tmfor the binding of the entire multi-part primer to the resultingcomplementary amplicon is 78.0° C.; the Tm for the binding of the24-14-5:1:1 anchor sequence to a template is 66.9° C., and the Tm forthe binding of the entire multi-part primer to the resultingcomplementary amplicon is 79.9° C.; and the Tm for the binding of the24-18-5:1:1 anchor sequence to a template is 67.9° C., and the Tm forthe binding of the entire multi-part primer to the resultingcomplementary amplicon is 79.3° C.

For each of the three multi-part primer designs, a series of PCRamplification and detection assays was carried out as described inExample 4, utilizing a dilution series starting with 10⁶ WT templatesplus 10⁶, 10⁵, 10⁴, 10³, 10², or 10¹ copies of the MUT template,respectively. The assay instrument automatically calculates thethreshold cycle (C_(T)) for each reaction. The C_(T) values calculatedfrom the real-time data for each reaction (not shown) are listed inTable 2, along with the calculated C_(T) value for reactions initiatedwith 10⁶ WT templates and no MUT templates.

TABLE 2 Threshold Cycles (C_(T)) Observed for Reactions ContainingDifferent Numbers of Intended Targets Primer 10⁶ 10⁵ 10⁴ 10³ 10² 10¹ 024-10-5:1:1 20.0 24.3 27.3 30.8 33.5 35.2 35.0 24-14-5:1:1 23.3 26.630.4 33.4 37.0 38.8 41.1 24-18-5:1:1 25.8 30.6 33.2 36.4 42.0 45.2 43.9

FIG. 11 is a set of graphs showing the C_(T) values observed (for eachset of reactions containing the same primer) as a function of thelogarithm of the number of MUT templates present in each reaction. Line1101 is a linear correlation fit to C_(T) values for the primer thatforms a bubble with a circumference that is 24-nucleotides long; line1102 is a linear correlation fit to C_(T) values for the primer thatforms a bubble with a circumference that is 32-nucleotides long; andline 1103 is a linear correlation fit to C_(T) values for the primerthat forms a bubble with a circumference that is 40-nucleotides long.Similar to what occurred with primers possessing longer foot sequences,when the 24-10-5:1:1 primer, which forms a relatively small bubble, wasused, the lower abundance MUT template samples gave C_(T) values thatoccurred somewhat earlier than predicted, suggesting the presence of afew obscuring amplicons generated from the abundant WT templates in thesample.

These results demonstrate that the use of a multi-part primer that formsa larger bubble, such as primer 24-14-5:1:1, reduces this problem, andthe use of a primer that forms the largest bubble, such as primer24-18-5:1:1, virtually eliminates this problem, enabling the detectionand quantitation of as few as 10 intended template molecules in thepresence of 1,000,000 unintended template molecules.

Example 7 EGFR Mutation L858R and the Effect of Varying the Position ofthe Interrogating Nucleotide within the Foot Sequence of a Multi-PartPrimer

The experiment described in Example 3 was repeated using the same24-14-5:1:1 primer (SEQ. ID No. 6) which includes aseven-nucleotide-long foot sequence in which the interrogatingnucleotide is located at the penultimate position from the 3′ end of theprimer, and also using five additional multi-part primers of the samedesign, except that the position of the interrogating nucleotide withthe foot sequence was varied. In all six cases, the anchor sequence was24-nucleotides long, the bridge sequence was 14-nucleotides long, andthe foot sequence was 7-nucleotides long. Primer sequences and theintended target sequence (MUT), were as follows:

Primer 24-14-6:1:0 Anchor Bridge Foot FP:  (SEQ ID No. 11)5′-ACTGGTGAAAACACCGCAGCATGTTGCACGAGTGAGCCTTGGGC G -3′ MUT: (SEQ ID No. 2) 3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAA CCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′Primer 24-14-5:1:1 Anchor Bridge Foot FP:  (SEQ ID No. 6)5′-CTGGTGAAAACACCGCAGCATGTCGCACGAGTGAGCCCTGGGC G G-3′ MUT: (SEQ ID No. 2) 3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAACCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′Primer 24-14-4:1:2 Anchor Bridge Foot FP:  (SEQ ID No. 12)5′-TGGTGAAAACACCGCAGCATGTCACACGAGTGAGCCACGGGC G G G-3′ MUT: (SEQ ID No. 2) 3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAACCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′Primer 24-14-3:1:3 Anchor Bridge Foot FP:  (SEQ ID No. 13)5′-GGTGAAAACACCGCAGCATGTCAAACGAGTGAGCCACAGGC G GG C-3′ MUT: (SEQ ID No. 2) 3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAACCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′Primer 24-14-2:1:4 Anchor Bridge Foot FP:  (SEQ ID No. 14)5′-GTGAAAACACCGCAGCATGTCAAGGAAGTGAGCCACAAGC G GGC C-3′ MUT: (SEQ ID No. 2) 3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAACCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′Primer 24-14-1:1:5 Anchor Bridge Foot FP:  (SEQ ID No. 15)5′-TGAAAACACCGCAGCATGTCAAGACAGACTGACCCAAAC G GGCC A-3′ MUT: (SEQ ID No. 2) 3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAACCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′ Reverse Primer RP: (SEQ ID No. 3) 5′-GCATGGTATTCTTTCTCTTCCGCA-3′

In the multi-part forward primers, the bridge sequence is underlined,and the interrogating nucleotide in the foot sequence is bolded,underlined, and larger. In the mutant target sequence, the bindingsequence for the forward primer's anchor and for the forward primer'sfoot are underlined, and the sequence of the reverse primer isunderlined. In addition, in the mutant target sequence, the nucleotidespecific to the mutant is bolded, underlined, and larger. UsingIntegrated DNA Technologies' SciTools program for calculating themelting temperatures of DNA hybrids (specifying parameters: [oligo]=0.06μM; [Na⁺]=60 mM; [Mg²⁺]=3 mM; [dNTPs]=0.25 mM); the Tm for the bindingof the 24-14-6:1:0 anchor sequence to a template is 67.9° C., and the Tmfor the binding of the entire multi-part primer to the resultingcomplementary amplicon is 79.0° C.; the Tm for the binding of the24-14-5:1:1 anchor sequence to a template is 66.9° C., and the Tm forthe binding of the entire multi-part primer to the resultingcomplementary amplicon is 79.9° C.; the Tm for the binding of the24-14-4:1:2 anchor sequence to a template is 68.1° C., and the Tm forthe binding of the entire multi-part primer to the resultingcomplementary amplicon is 80.0° C.; the Tm for the binding of the24-14-3:1:3 anchor sequence to a template is 67.0° C., and the

Tm for the binding of the entire multi-part primer to the resultingcomplementary amplicon is 78.9° C.; the Tm for the binding of the24-14-2:1:4 anchor sequence to a template is 65.6° C., and the Tm forthe binding of the entire multi-part primer to the resultingcomplementary amplicon is 78.2° C.; and the Tm for the binding of the24-14-1:1:5 anchor sequence to a template is 66.6° C., and the Tm forthe binding of the entire multi-part primer to the resultingcomplementary amplicon is 78.1° C.

PCR amplifications were carried out as described in Example 1. Real-timefluorescence results, that is, SYBR Green® fluorescence intensity as afunction of the number of amplification cycles completed, are shown inthe six panels of FIG. 12, where each panel identifies the multi-partprimer that was used. In each panel the odd-numbered curve is theresults obtained for a sample begun containing 10⁶ MUT templates, andthe even-numbered curve is the results obtained for a sample containing10⁶ WT templates. Table 3 lists the machine-calculated C_(T) values forboth targets with each primer, and also shows the difference (ΔC_(T)).

TABLE 3 Threshold Cycles (C_(T)) Observed for Reactions ContainingPrimers whose Interrogating Nucleotide is Located at Different Positionsin the Foot Sequence Primer 10⁶ MUT Templates 10⁶ WT Templates ΔC_(T)24-14-6:1:0 24.3 43.1 18.8 24-14-5:1:1 22.9 41.1 18.2 24-14-4:1:2 21.236.1 14.9 24-14-3:1:3 23.0 35.2 12.2 24-14-2:1:4 23.1 33.2 10.124-14-1:1:5 21.1 30.4 9.3

Example 8 EGFR Mutation L858R and the Effect of Varying Multi-PartPrimer Bubble Symmetry

The experiment described in Example 3 was repeated using the same24-14-5:1:1 primer (SEQ. ID No. 6), which forms a symmetrical bubblethat includes its 14-nucleotide-long bridge sequence and a14-nucleotide-long intervening sequence from the template; and theexperiment also used four additional multi-part primers that formdifferent asymmetric bubbles with the mutant target (SEQ ID No. 2). By“asymmetric bubble,” we mean a bubble formed by a bridge sequence and anintervening sequence in the template that have different lengths. Inthis experiment, all of the multi-part primers that were compared had ananchor sequence 24-nucleotides long, a 5:1:1 foot sequence, and adifferent-length bridge sequence (which were 18, 16, 14, 12, or 10nucleotides in length). For each multi-part primer, the identity of theanchor sequence was selected so that the sum of the length of the bridgesequence plus the length of the intervening sequence (formed by thebinding of both the anchor sequence and the foot sequence to thetemplate) equals 28. Consequently, the circumference of the bubbleformed by each of these five multi-part primers was always the same. Theaim of the experiment was to determine whether or not the formation ofan asymmetrical bubble affects the selectivity of the primer. Primersequences and the intended target sequence (MUT) were as follows:

Primer 24-18/10-5:1:1 Anchor Bridge Foot FP:  (SEQ ID No. 16)5′-TGAAAACACCGCAGCATGTCAAGACACACGACAAGTGAGCCCTGGGC G G-3′ MUT: (SEQ ID No. 2) 3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAACCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′Primer 24-16/12-5:1:1 Anchor Bridge Foot FP:  (SEQ ID No. 17)5′-GGTGAAAACACCGCAGCATGTCAATCCAACAAGTGAGCCCTGGGC G G-3′ MUT: (SEQ ID No. 2) 3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAACCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′Primer 24-14/14-5:1:1 Anchor Bridge Foot FP:  (SEQ ID No. 6)5′-CTGGTGAAAACACCGCAGCATGTCGCACGAGTGAGCCCTGGGC G G-3′ MUT: (SEQ ID No. 2) 3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAACCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′Primer 24-12/16-5:1:1 Anchor Bridge Foot FP:  (SEQ ID No. 18)5′-TACTGGTGAAAACACCGCAGCATGGACGACGAGCCCTGGGC G G-3′ MUT:  (SEQ ID No. 2)3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAACCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′Primer 24-10/18-5:1:1 Anchor Bridge Foot FP:  (SEQ ID No. 19)5′-CGTACTGGTGAAAACACCGCAGCACTGACGGCCCTGGGC G G-3′ MUT:  (SEQ ID No. 2)3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAACCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′ Reverse Primer RP:(SEQ ID No. 3) 5′-GCATGGTATTCTTTCTCTTCCGCA-3′

In the multi-part forward primers, the bridge sequence is underlined,and the interrogating nucleotide in the foot sequence is bolded,underlined, and larger. In the mutant target sequence, the bindingsequence for the forward primer's anchor and for the forward primer'sfoot are underlined, and the sequence of the reverse primer isunderlined. In addition, in the mutant target sequence, the nucleotidespecific to the mutant is bolded, underlined, and larger. UsingIntegrated DNA Technologies' SciTools program for calculating themelting temperatures of DNA hybrids (specifying parameters: [oligo]=0.06μM; [Na⁺]=60 mM; [Mg²⁺]=3 mM; [dNTPs]=0.25 mM); the Tm for the bindingof the 24-18/10-5:1:1 anchor sequence to a template is 66.3° C., and theTm for the binding of the entire multi-part primer to the resultingcomplementary amplicon is 79.1° C.; the Tm for the binding of the24-16/12-5:1:1 anchor sequence to a template is 67.0° C., and the Tm forthe binding of the entire multi-part primer to the resultingcomplementary amplicon is 78.5° C.; the Tm for the binding of the24-14/14-5:1:1 anchor sequence to a template is 66.9° C., and the Tm forthe binding of the entire multi-part primer to the resultingcomplementary amplicon is 79.9° C.; the Tm for the binding of the24-12/16-5:1:1 anchor sequence to a template is 66.3° C., and the Tm forthe binding of the entire multi-part primer to the resultingcomplementary amplicon is 79.5° C.; and the Tm for the binding of the24-10/18-5:1:1 anchor sequence to a template is 67.9° C., and the Tm forthe binding of the entire multi-part primer to the resultingcomplementary amplicon is 79.3° C.

PCR amplifications were carried out as described in Example 1. Real-timefluorescence results, that is, SYBR Green® fluorescence intensity as afunction of the number of amplification cycles completed are shown inthe five panels of FIG. 13, where each panel identifies the bubble thebubble that can be formed by the length of the bridge sequence and thelength of the target's intervening sequence (for example, an “18/10Bubble” signifies use of forward primer 24-18/10-5:1:1 which can form anintervening sequence with the target that is 10 nucleotides long). Ineach panel the odd-numbered curve is the results obtained for a samplebegun containing 10⁶ MUT templates, and the even-numbered curve is theresults obtained for a sample containing 10⁶ WT templates. Table 4 liststhe machine-calculated C_(T) values for both targets with each primer,and also shows the difference (ΔC_(T)).

TABLE 4 Threshold Cycles (C_(T)) Observed for Reactions ContainingPrimers that Form Bubbles with Varying Symmetries Primer 10⁶ MUTTemplates 10⁶ WT Templates ΔC_(T) 24-18/10-5:1:1 22.8 39.3 16.524-16/12-5:1:1 22.1 38.2 16.1 24-14/14-5:1:1 22.9 41.1 18.224-12/16-5:1:1 22.5 38.4 15.9 24-10/18-5:1:1 22.1 39.5 17.4

Example 9 B-raf Mutation V600E

We used the method of Example 4 with a multi-part primer according tothis invention targeted to B-raf mutation V600E, which is asingle-nucleotide polymorphism. For comparative purposes, we utilized a24-14-5:1:1 design for the primer. The primer sequences and the intendedtarget sequence (MUT) were as follows:

B-raf Primer Anchor Bridge Foot FP:  (SEQ ID No. 20)5′-AGACAACTGTTCAAACTGATGGGAAAACACAATCATCTATTTC T C-3′ MUT: (SEQ ID No. 21) 3′-GGTCTGTTGACAAGTTTGACTACCCTGGGTGAGGTAGCTCTAAAGAGACATCGATCTGGTTTTAGTGGATAAAAA-5′ Reverse Primer RP:  (SEQ ID No. 22)5′-ATAGGTGATTTTGGTCTAGC-3′

In the multi-part forward primer, the bridge sequence is underlined, andthe interrogating nucleotide in the foot sequence is bolded, underlined,and larger. In the mutant target sequence, the binding sequence for theforward primer's anchor and the binding sequence for the forwardprimer's foot are underlined, and the sequence of the reverse primer isunderlined. Using Integrated DNA Technologies' SciTools program forcalculating the melting temperatures of DNA hybrids (specifyingparameters: [oligo]=0.06 μM; [Na⁺]=60 mM; [Mg²⁺]=3 mM; [dNTPs]=0.25 mM),the Tm for the binding of the anchor sequence to a template is 63.5° C.,the Tm for the binding of the entire multi-part primer to the resultingcomplementary amplicon is 71.1° C., and the calculated Tm for thebinding of the reverse primer is 56.1° C.

Plasmids were prepared by inserting synthetic oligonucleotides into apGEM-11Zf(+) vector (Promega) that corresponded to a 116 bp EGFR genefragment that contained either the B-raf V600E mutation or the B-rafwild-type sequence. Mutant and wild-type plasmid DNA was digested withrestriction endonuclease Mse I (New England Biolabs). The digestionmixture contained 10 units Mse I and 4 μg of mutant or wild-type genomicDNA in a 20-μl volume that contained 5 mM KAc, 2 mM Tris-Ac (pH 7.9), 1mM MgAc, 1% bovine serum albumin, and 100 μM dithiothreitol. Thereactions were incubated for 120 min at 37° C., followed by anincubation for 20 min at 65° C. to inactivate the enzyme.

PCR amplifications were performed in a 30-μl volume containing 50 mMKCl, 10 mM Tris-HCl (pH 8.0), 3 mM MgCl₂, 1.5 Units AmpliTaq Gold DNApolymerase, 250 μM of each deoxyribonucleoside triphosphate (dNTP), 60nM of each primer, and 1×SYBR® Green. Amplifications were carried outusing 0.2 ml polypropylene PCR tubes (white) in a Bio-Rad IQ5spectrofluorometric thermal cycler. The thermal-cycling profile was 10min at 95° C., followed by 60 cycles of 94° C. for 15 sec, 60° C. for 20sec, and 72° C. for 20 sec. SYBR® Green fluorescence intensity wasmeasured at the end of each chain elongation stage (72° C.).

The PCR amplification and detection assays were carried out, utilizing adilution series containing 10⁶ WT templates plus 10⁶, 10⁵, 10⁴, 10³,10², or 10¹ copies of the MUT template, respectively. We also included asample containing only 10⁶ WT templates. From the real-time fluorescencedata (not shown), the assay instrument automatically calculates thethreshold cycle (C_(T)) for each reaction. For the B-raf V600E mutantdilution series, those values were 27.7 (10⁶ MUT templates), 31.1 (10⁵MUT templates), 34.1 (10⁴ MUT templates), 37.6 (10³ MUT templates), 43.0(10² MUT templates), 46.9 (10′ MUT templates), and 50.8 (10⁶ WTtemplates and no MUT templates). FIG. 14 is a graph of the C_(T) valueobserved for each reaction that contained MUT templates, as a functionof the logarithm of the number of MUT templates present in thatreaction. Line 1401 is a linear correlation fit to the data points.Dashed line 1402 identifies the C_(T) value for the amplificationinitiated with 10⁶ WT templates and no MUT templates.

Example 10 EGFR Mutation T790M in Human Genomic DNA

A series of PCR amplification and detection assays was carried out usingas templates human genomic DNA containing EGFR mutation T790M (isolatedfrom cell line H1975, which contains the EFGR T790M mutation) and humangenomic DNA containing the corresponding wild-type sequence (isolatedfrom human genomic DNA obtained from Coriell Cell Repositories), whichdiffer by a single-nucleotide polymorphism in the EGFR gene. The forwardprimer was a 24-14-4:1:1 multi-part primer according to this invention.The reverse primer was a conventional linear primer. The primersequences and the intended target sequence (MUT) were as follows:

T790M Primer Anchor Bridge Foot FP:  (SEQ ID No. 23)5′-GCCTGCTGGGCATCTGCCTCACCTAATAATCTACAACAATCA T G-3′ MUT: (SEQ ID No. 24) 3′-CACGGCGGACGACCCGTAGACGGAGTGGAGGTGGCACGTCGAGTAGTACGTCGAGTACGGGAAGCCGACGGAGGACC-5′ Reverse Primer RP:  (SEQ ID No. 25)5′-GAGGCAGCCGAAGGGCATGAGC-3′

In the multi-part forward primer, the bridge sequence is underlined, andthe interrogating nucleotide in the foot sequence is bolded, underlined,and larger. In the mutant target sequence, the binding sequence for theforward primer's anchor and the binding sequence for the forwardprimer's foot are underlined, and the sequence of the reverse primer isunderlined. Using Integrated DNA Technologies' SciTools program forcalculating the melting temperatures of DNA hybrids (specifyingparameters: [oligo]=0.06 μM; [Na⁺]=60 mM; [Mg²⁺]=3 mM; [dNTPs]=0.25 mM),the Tm for the binding of the anchor sequence to a template is 72.5° C.,the Tm for the binding of the entire multi-part primer to the resultingcomplementary amplicon is 73.9° C., and the calculated Tm for thebinding of the reverse primer is 68.2° C.

Mutant and wild-type human genomic DNAs were digested with restrictionendonuclease Mse I. The digestion mixture contained 10 units Mse I and 4μg of mutant or wild-type genomic DNA in a 20-μl volume that contained 5mM KAc, 2 mM Tris-Ac (pH 7.9), 1 mM MgAc, 1% bovine serum albumin, and100 μM dithiothreitol. The reactions were incubated for 120 min at 37°C., followed by incubation for 20 min at 65° C. to inactivate theenzyme.

PCR amplifications were performed in a 20-μl volume containing 50 mMKCl, 10 mM Tris-HCl (pH 8.0), 3 mM MgCl₂, 1.0 Unit AmpliTaq Gold DNApolymerase, 250 μM of each deoxyribonucleoside triphosphate (dNTP), 60nM of each primer, and 1×SYBR® Green. Amplifications were carried outusing 0.2 ml polypropylene PCR tubes (white) on a Bio-Rad IQ5spectrofluorometric thermal cycler. The thermal-cycling profile was 10min at 95° C., followed by 60 cycles of 94° C. for 15 sec, 55° C. for 15sec, and 72° C. for 20 sec. SYBR® Green fluorescence intensity wasmeasured at the end of each chain elongation stage (72° C.).

The PCR amplification and detection assays were carried out, utilizing adilution series containing 10,000 WT templates plus: 10,000; 3,000;1,000; 300; 100; 30; or 10 copies of the MUT template, respectively. Wealso included a sample containing only 10,000 WT templates. From thereal-time fluorescence data (not shown), the assay instrumentautomatically calculates the threshold cycle (C_(T)) for each reaction.For this T790M dilution series, those values were 29.2 (10,000 MUTtemplates), 31.1 (3,000 MUT templates), 32.7 (1,000 MUT templates), 35.5(300 MUT templates), 38.2 (100 MUT templates), 38.8 (30 MUT templates),40.7 (10 MUT templates), and 42.8 (10,000 WT templates and no MUTtemplates). FIG. 15 is a graph of the C_(T) value observed for eachreaction that contained MUT templates, as a function of the logarithm ofthe number of MUT templates present in that reaction. Line 1501 is alinear correlation fit to the data points. Dashed line 1502 is the C_(T)value for the amplification initiated with 10,000 WT templates and noMUT templates.

Example 11 EGFR Mutation L858R Quantitated in the Applied BiosystemsPRISM 7700 Spectrofluorometric Thermal Cycler

An experiment similar to the assay reported in Example 4 was performedto amplify and detect mutation L858R in the EGFR gene, utilizing adifferent thermal cycling instrument, the Applied Biosystems PRISM 7700spectrofluorometric thermal cycler. A series of PCR amplification anddetection assays was carried out using as templates plasmid DNAcontaining EGFR mutation L858R and plasmid DNA containing thecorresponding wild-type sequence, which differ by a single-nucleotidepolymorphism in the EGFR gene. In contrast to the templates used inExample 4, in this experiment, the templates were not digested with arestriction endonuclease. The amplifications were carried out with thesame multi-part forward primer and conventional reverse primer asdescribed in Example 3. The primer sequences and the intended targetsequence (MUT) were as follows:

Primer 24-14-5:1:1 Anchor Bridge Foot FP:  (SEQ ID No. 6)5′-CTGGTGAAAACACCGCAGCATGTCGCACGAGTGAGCCCTGGGC G G-3′ MUT: (SEQ ID No. 2) 3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAACCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′ RP:  (SEQ ID No. 3)5′-GCATGGTATTCTTTCTCTTCCGCA-3′

In the multi-part forward primer, the bridge sequence is underlined, andthe interrogating nucleotide in the foot sequence is bolded, underlined,and larger. In the mutant target sequence, the binding sequence for theforward primer's anchor and the binding sequence for the forwardprimer's foot are underlined, and the sequence of the reverse primer isunderlined. Using Integrated DNA Technologies' SciTools program forcalculating the melting temperatures of DNA hybrids (specifyingparameters: [oligo]=0.06 μM; [Na⁺]=60 mM; [Mg²⁺]=3 mM; [dNTPs]=0.25 mM),the Tm for the binding of the anchor sequence to a template is 66.9° C.,the Tm for the binding of the entire multi-part primer to the resultingcomplementary amplicon is 79.9° C., and the calculated Tm for thebinding of the reverse primer is 68.2° C.

PCR amplifications were performed in a 40-μl volume that contained 50 mMKCl, 10 mM Tris-HCl (pH 8.0), 3 mM MgCl₂, 2.0 Units AmpliTaq Gold DNApolymerase, 250 μM of each deoxyribonucleoside triphosphate (dNTP), 60nM of each primer, and 1×SYBR® Green. Amplifications were carried outusing 0.2 ml polypropylene PCR tubes (transparent) on the AppliedBiosystems PRISM 7700 spectrofluorometric thermal cycler. Thethermal-cycling profile was 10 min at 95° C., followed by 55 cycles of94° C. for 15 sec, 60° C. for 20 sec, and 72° C. for 20 sec. SYBR® Greenfluorescence intensity was measured at the end of each chain elongationstage (72° C.).

The PCR amplification and detection assays were carried out, utilizing adilution series containing 10⁶ WT templates plus 10⁶, 10⁵, 10⁴, 10³,10², or 10¹ copies of the MUT template, respectively. We also included asample containing only 10⁶ WT templates. From the real-time fluorescencedata (not shown), the assay instrument automatically calculates thethreshold cycle (C_(T)) for each reaction. Those values were 21.2 (10⁶MUT templates), 24.9 (10⁵ MUT templates), 28.3 (10⁴ MUT templates), 32.2(10³ MUT templates), 36.0 (10² MUT templates), 37.6 (10¹ MUT templates)and 38.7 (10⁶ WT templates and no MUT templates). FIG. 16 is a graph ofthe C_(T) value observed for each reaction that contained MUT templates,as a function of the logarithm of the number of MUT templates present inthat reaction. Line 1601 is a linear correlation fit to the data points.Dashed line 1602 is the C_(T) value for the amplification initiated with10⁶ WT templates and no MUT templates.

Example 12 Role of ARMS Discrimination when Multi-Part Primers areUtilized in PCR Assays

To investigate the functioning of multi-part primers according to thisinvention, we repeated the experiment described in Example 3, not onlywith the 24-14-5:1:1 primer described there, but also with a truncated24-14-5:0:0 primer, that is a primer that had the same anchor sequence,the same bridge sequence and the same five 5′ nucleotides of the footsequence. It lacked the last two 3′ nucleotides of the foot sequence.Thus, its foot sequence was perfectly complementary to both theintended, mutant target, and the unintended, wild-type target. Primersequences and the intended target sequence (MUT), were as follows forreactions utilizing each of these two multi-part primers:

Primer 24-14-5:1:1 Anchor Bridge Foot FP:  (SEQ ID No. 6)5′-CTGGTGAAAACACCGCAGCATGTCGCACGAGTGAGCCCTGGGC G G-3′ MUT: (SEQ ID No. 2) 3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAACCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′Primer 24-14-5:0:0 Anchor Bridge Foot FP:  (SEQ ID No. 26)5′-CTGGTGAAAACACCGCAGCATGTCGCACGAGTGAGCCCTGGGC-3′ MUT:  (SEQ ID No. 2)3′-CCTTGCATGACCACTTTTGTGGCGTCGTACAGTTCTAGTGTCTAAAA CCCGCCCGGTTTGACGACCCACGCCTTCTCTTTCTTATGGTACGTCT T-5′ Reverse Primer RP: (SEQ ID No. 3) 5′-GCATGGTATTCTTTCTCTTCCGCA-3′

In the multi-part forward primers, the bridge sequence is underlined,and the interrogating nucleotide in the foot sequence is bolded,underlined, and larger. In the mutant target sequence, the bindingsequence for the forward primer's anchor and the binding sequence forthe forward primer's foot are underlined, and the sequence of thereverse primer is underlined. Using Integrated DNA Technologies'SciTools program for calculating the melting temperatures of DNA hybrids(specifying parameters: [oligo]=0.06 μM; [Na⁺]=60 mM; [Mg²⁺]=3 mM;[dNTPs]=0.25 mM), the Tm for the binding of the anchor sequence of bothprimers to a template is 66.9° C., the Tm for the binding of primer24-14-5:1:1 to the resulting complementary amplicon is 79.9° C., and theTm for the binding of primer 24-14-5:0:0 to the resulting complementaryamplicon is 79.0° C.

PCR amplifications were carried out as described in Example 3. Real-timefluorescence results, that is, SYBR Green® fluorescence intensity as afunction of the number of amplification cycles completed were recordedfor each reaction. FIG. 17, Panel A shows the results obtained forreactions containing primer 24-14-5:1:1, where curve 1701 is thereaction containing 10⁶ MUT templates and curve 1702 is the reactioncontaining 10⁶ WT templates; and FIG. 17, Panel B shows the resultsobtained for reactions containing primer 24-14-5:0:0, where curve 1703is the reaction containing 10⁶ MUT templates and curve 1704 is thereaction containing 10⁶ WT templates. The assay instrument automaticallycalculates the threshold cycle (C_(T)) for each curve. The C_(T) valuesfor primer 24-14-5:1:1 were 23.1 (curve 1701) and 40.7 (curve 1702),giving a ΔC_(T) of 17.6 cycles; and the C_(T) values for primer24-14-5:0:0 were 39.7 (curve 1703) and 39.4 (curve 1704), giving aΔC_(T) of −0.3 cycles (indicating that these two reactions gavevirtually identical results).

The foregoing examples and description of the preferred embodimentsshould be taken as illustrating, rather than as limiting the presentinvention as defined by the claims. As will be readily appreciated,numerous variations and combinations of the features set forth above canbe utilized without departing from the present invention as set forth inthe claims. Such variations are not regarded as a departure from thescope of the invention, and all such variations are intended to beincluded within the scope of the following claims. All references citedherein are incorporated by reference in their entireties.

1. A primer-dependent nucleic acid amplification method having aninitial primer-annealing temperature capable of amplifying at least onerare, intended nucleic acid target sequence in an amplification reactionmixture containing, for each intended target sequence, an abundant,closely related unintended nucleic acid target sequence where theintended target sequence includes a nucleotide substitution (asingle-nucleotide polymorphism) from the unintended target sequence,comprising repeatedly cycling a reaction mixture comprising the targetsequences and the other reagents needed for amplification including aDNA polymerase and, for each intended target sequence, a primer pairthat includes a multi-part primer having, in the direction 5′ to 3′, ananchor sequence, a bridge sequence and a foot sequence, wherein: (a) theanchor sequence hybridizes to a binding site in the intended andunintended target sequences at the primer-annealing temperature; (b) thefoot sequence consists of 5-8 nucleotides that are perfectlycomplementary to the intended target sequence at a location that isseparated from the anchor-sequence binding site by an interveningsequence, and that includes an interrogating nucleotide complementary tothe single-nucleotide polymorphism present in the intended targetsequence, and that hybridizes to the intended target sequence and isextended by the DNA polymerase; and (c) when the anchor and footsequences are hybridized to the intended target sequence, the bridgesequence in the primer and the intervening sequence in the intendedtarget sequence form an unhybridized bubble 20-52 nucleotides incircumference; and wherein the probability of the unintended targetbeing copied by the DNA polymerase is at least 1,000 times less than theprobability of the intended target sequence being copied. 2-23.(canceled)